CN111936519A - Antibodies specific for CD70 and uses thereof - Google Patents

Abstract

The present invention provides antibodies that specifically bind to CD70 (cluster of differentiation 70). The invention also provides bispecific antibodies that bind to CD70 and another antigen (e.g., CD 3). The invention also relates to nucleic acids encoding the antibodies, and methods of obtaining such antibodies (monospecific and bispecific). The invention also relates to therapeutic methods of using these antibodies to treat CD 70-mediated conditions, including cancer.

Description

Antibodies specific for CD70 and uses thereof

Cross Reference to Related Applications

This application claims priority from U.S. provisional patent application No. 62/641,873 filed on 12.3.2018 and U.S. provisional patent application No. 62/625,019 filed on 1.2.2018, each of which is incorporated herein by reference in its entirety.

Reference to sequence listing

This application is being submitted electronically via the EFS-Web and includes a sequence listing submitted electronically in the txt format. The txt file contains a sequence list entitled "ALGN-015 _02WO _ SL. txt" created on day 1, month 3, 2019 and 234,861 bytes in size. The sequence listing contained in the txt file is part of the specification and is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to antibodies, e.g., full length antibodies or antigen binding fragments thereof, that specifically bind to cluster of differentiation 70(CD 70). The invention also relates to heteromultimeric antibodies (e.g., bispecific antibodies) comprising the CD70 antibody on one arm. Also provided are compositions comprising the CD70 antibodies, methods of producing and purifying such antibodies, and their use in diagnosis and therapy.

Background

Renal Cell Carcinoma (RCC) is a cancer originating in the renal cortex, accounting for about 90% of kidney cancers. According to histology, RCC can be divided into several subtypes, with renal clear cell carcinoma (ccRCC) being the most common and leading to the most death. Each year, over 320,000 RCCs are reported worldwide, resulting in approximately 140,000 deaths. The incidence of RCC has steadily increased over the last 10 years, accounting for 2-3% of all adult malignancies. Patients with early stage local tumor can choose to be removed by operation; however, local disease may experience early hematogenous dissemination, leading to metastasis. Sites of early metastasis include lung, lymph node, liver, bone and brain; the adrenal glands and contralateral kidneys are less frequent. Patients with advanced disease face high morbidity with a 5-year median survival rate of 53% for stage III disease, and only 8% for metastatic disease. Current first-line treatment options for advanced disease include small molecule Tyrosine Kinase Inhibitors (TKIs) targeting Vascular Endothelial Growth Factor (VEGF) receptors, such as sunitinib and pazopanib, monoclonal antibodies targeting VEGF, such as bevacizumab, the mammalian target of rapamycin (mTOR) inhibitor temsirolimus, and high doses of IL-2. Although these VEGF-targeted therapies improve overall survival, long-term resistance can lead to disease recurrence and treatment of advanced disease is still not satisfactory (see, e.g., zararabi, k. et al, Journal of Hematology and Oncology,10:38 (2017)).

Cluster of differentiation 70(CD70, CD27LG, or TNFSF7) is a member of the Tumor Necrosis Factor (TNF) superfamily and is also a ligand for the TNF superfamily receptor CD 27. Transient interactions between CD27 and CD70 provide complementary T cell co-stimulation as provided by CD 28. CD70 is expressed on hematological cancers (such as non-hodgkin lymphoma and hodgkin's disease) as well as solid tumors (such as glioblastoma and renal cell carcinoma); its expression on ccRCC is almost uniform (see, e.g., Grewal I. et al, Expert Opinion on Therapeutic Targets,12(3):341- "351 (2008)).

Recently, CD70 bispecific antibodies have been developed in the form of T cell engaging bispecific approaches. However, many bispecific formats are limited in their small molecular weight and short half-life, thus requiring continuous infusion. Thus, there remains a need for antibodies (e.g., monospecific or bispecific antibodies) with improved efficacy and safety profiles and suitable for use in human patients for the treatment of CD 70-expressing cancers, particularly mRCC.

Disclosure of Invention

The invention disclosed herein relates to antibodies (e.g., monospecific or bispecific antibodies) that specifically bind to cluster of differentiation 70(CD 70). In some embodiments, the full-length bispecific form of the CD70 antibody as described herein has a longer half-life, minimized Fc interactions, and minimized non-specific cytokine release in vivo via interaction with immune cells.

Accordingly, in one aspect, the invention provides an isolated antibody that specifically binds to CD70, wherein the antibody comprises: (a) a heavy chain Variable (VH) region comprising: (i) VH complementarity determining region 1(CDR1) comprising SEQ ID NOs 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 406, 368, 369, 370, 368, 375, 392, 380, 376, 382, 398, 393, 410, 416, 220, 2, 382, 393, 2, 393, 410, 393, and 393, 410, 422. 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459, or 460; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 449, 450, 455, 456, 461, or 462; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457 or 463; and/or a light chain Variable (VL) region comprising: (i) a VL CDR1 comprising the sequence set forth in SEQ ID NO 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, or 527; (ii) a VL CDR2 comprising a sequence set forth in SEQ ID NO 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, or 528; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526 or 529.

In another aspect, there is provided an isolated antibody that specifically binds to CD70, wherein the antibody comprises: a VH region comprising a VH CDR1, VH CDR2 and VH CDR3 of the VH sequence set forth in SEQ ID NOs 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329 or 331; and/or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NOs 1,3, 5,7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, or 330. In some embodiments, a VH region as described herein comprises variants in which one or several conservative amino acid substitutions are located in residues that are not within a CDR, and/or a VL region as described herein comprises variants in which one or several amino acid substitutions are located in amino acids that are not within a CDR. For example, in some embodiments, the VH or VL region may comprise an amino acid sequence described above or a variant thereof in which no more than 10, 9,8, 7, 6,5, 4, 3,2, or 1 conservative substitutions are located in residues that are not within a CDR.

In some embodiments, there is provided an isolated antibody that specifically binds to CD70, wherein the antibody comprises: a VH region comprising the sequence shown in SEQ ID NO 18; and/or a VL domain comprising the sequence shown in SEQ ID NO 17.

In some embodiments, an isolated antibody that specifically binds to CD70 and competes with an isolated antibody provided herein that specifically binds to CD70 is provided.

In another aspect, a bispecific antibody is provided, wherein the bispecific antibody is a full length antibody comprising a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen (e.g., CD70), and a second antibody variable domain of the bispecific antibody that is capable of recruiting the activity of a human immune effector cell by specifically binding to an effector antigen (e.g., cluster of differentiation 3(CD3)) that is localized on the human immune effector cell. In some embodiments, the first antibody variable domain comprises a heavy chain Variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NOs 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, or 331; and/or a light chain Variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NOs 1,3, 5,7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, or 330. In some embodiments, the first antibody variable domain comprises (a) a heavy chain Variable (VH) region comprising: (i) VH complementarity determining region 1(CDR1) comprising SEQ ID NOs 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 406, 368, 369, 370, 368, 375, 392, 380, 376, 382, 398, 393, 410, 416, 220, 2, 382, 393, 2, 393, 410, 393, and 393, 410, 422. 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459, or 460; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 449, 450, 455, 456, 461, or 462; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457 or 463; and/or (b) a light chain Variable (VL) region comprising: (i) a VL CDR1 comprising the sequence set forth in SEQ ID NO 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, or 527; (ii) a VL CDR2 comprising a sequence set forth in SEQ ID NO 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, or 528; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526 or 529.

In some embodiments, the second antibody variable domain comprises VH and/or VL regions specific for CD 3. For example, the second antibody variable domain comprises a heavy chain Variable (VH) region comprising the VH CDR1, VH CDR2 and VH CDR3 of the VH sequence shown in SEQ ID NO: 266; and/or a light chain variable region (VL) comprising the VL CDR1, VL CDR2 and VL CDR3 of the VL sequence shown in SEQ ID NO: 265. In some embodiments, the second antibody variable domain comprises (a) a VH region comprising (i) a VH CDR1 comprising the sequence shown in SEQ ID NOs 267, 268, or 269; (ii) VH CDR2 comprising the sequence shown in SEQ ID NO 270 or 271; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 272; and/or a VL region comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 273; (ii) VL CDR2 comprising the sequence shown in SEQ ID NO. 274; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 275.

In some embodiments, an antibody described herein comprises a constant region. In some embodiments, the antibodies described herein are antibodies of the human IgG1, IgG2, or IgG2 Δ a, IgG3, or IgG4 subclasses. In some embodiments, the antibodies described herein comprise a glycosylated constant region. In some embodiments, an antibody described herein comprises a constant region having reduced binding affinity for one or more human fey receptors.

In some embodiments, both the first antibody variable domain and the second antibody variable domain of the bispecific antibody comprise amino acid modifications at positions 223, 225, and 228 (e.g., (C223E or C223R), (E225R), and (P228E or P228R)) in the hinge region of human IgG2(SEQ ID NO:279), and amino acid modifications at positions 409 or 368 in the CH3 region (e.g., K409R or L368E (EU numbering scheme)).

In some embodiments, both the first antibody variable domain and the second antibody variable domain of the bispecific antibody comprise an amino acid modification at position 265 of human IgG2 (e.g., D265A).

In some embodiments, both the first antibody variable domain and the second antibody variable domain of the bispecific antibody comprise an amino acid modification at one or more of positions 265 (e.g., D265A), 330 (e.g., a330S), and 331 (e.g., P331S) of human IgG 2. In some embodiments, both the first antibody variable domain and the second antibody variable domain of the bispecific antibody comprise an amino acid modification at each of positions 265 (e.g., D265A), 330 (e.g., a330S), and 331 (e.g., P331S) of human IgG 2.

In other embodiments, the invention provides pharmaceutical compositions comprising any of the antibodies described herein.

The invention also provides cell lines that recombinantly produce any of the antibodies described herein.

The invention also provides nucleic acids encoding any of the antibodies described herein. The invention also provides a nucleic acid encoding the heavy chain variable region and/or the light chain variable region of any of the antibodies described herein.

The invention also provides a host cell comprising a nucleic acid or vector provided herein. Also provided is a method of producing an antibody (e.g., a monospecific or bispecific antibody) provided herein, comprising culturing a host cell provided herein under conditions that result in the production of the antibody, and isolating the antibody from the host cell or culture.

The invention also provides kits comprising an effective amount of any of the antibodies or antibody conjugates described herein.

Also provided is an antibody or bispecific antibody provided herein for use as a medicament.

The invention also provides a method of treating a subject in need thereof, the method comprising providing an isolated antibody or bispecific antibody described herein, and administering the antibody to the subject.

Also provided are methods of treating a disorder associated with malignant cells expressing CD70 in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an antibody as described herein. In some embodiments, the disorder is cancer. In some embodiments, the cancer is a CD 70-associated cancer (e.g., any cancer with CD70 expression) selected from the group consisting of: renal cell carcinoma, glioblastoma, gliomas such as low grade glioma, non-hodgkin's lymphoma (NHL), Hodgkin's Disease (HD), waldenstrom's macroglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

In another aspect, the invention provides a method of inhibiting tumor growth or progression in a subject having malignant cells that express CD70, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an isolated antibody or bispecific antibody as described herein.

In another aspect, the invention provides a method of inhibiting metastasis of a CD 70-expressing malignant cell in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an isolated antibody or bispecific antibody as described herein.

In another aspect, the invention provides a method of causing tumor regression in a subject having malignant cells expressing CD70, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an isolated antibody or bispecific antibody as described herein.

Detailed Description

The invention disclosed herein provides antibodies (e.g., monospecific or bispecific antibodies) that specifically bind to CD70 (e.g., human CD 70). The invention also provides polynucleotides encoding these antibodies, compositions comprising these antibodies, and methods of making and using these antibodies. The invention also provides methods of treating a disorder associated with a CD 70-mediated condition in a subject, such as cancer. In particular, the inventors of the present invention found that the full-length bispecific form of CD70 antibody as described herein has a longer half-life, minimized Fc interactions and minimized non-specific cytokine release in vivo via interaction with immune cells.

General techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, virology, monoclonal antibody production and engineering, which are within the skill of the art. Such techniques are explained fully in the following references, such as Molecular Cloning, A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; oligonucleotide Synthesis (m.j. gait eds., 1984); methods in Molecular Biology, human Press; cell Biology A Laboratory Notebook (J.E.Cellis eds., 1998) Academic Press; animal Cell Culture (r.i. freshney eds, 1987); introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts,1998) Plenum Press; cell and Tissue Culture Laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell eds., 1993-1998) J.Wiley and Sons; methods in Enzymology (Academic Press, Inc.); handbook of Experimental Immunology (eds. d.m.weir and c.c.blackwell); gene Transfer Vectors for Mammalian Cells (eds. J.M.Miller and M.P.Calos, 1987); current Protocols in Molecular Biology (ed. F.M. Ausubel et al, 1987); PCR The Polymerase Chain Reaction, (Mullis et al eds., 1994); current Protocols in Immunology (J.E. Coligan et al, 1991); short Protocols in Molecular Biology (Wiley and Sons, 1999); immunobiology (c.a. janeway and p.travers, 1997); antibodies (p.finch, 1997); antibodies a practical prophach (D.Catty. eds., IRL Press, 1988-1989); monoclonal antigens a practical proproach (edited by P.Shepherd and C.dean, Oxford University Press, 2000); using Antibodies a Laboratory manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M.Zantetti and J.D.Capra, eds., Harwood Academic Publishers, 1995).

Definition of

An "antibody" is an immunoglobulin molecule capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site that is localized in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab ', F (ab')₂Fv), single chain (ScFv) and domain antibodies (including, for example, shark and camelid antibodies), and fusion proteins comprising the antibodies, as well as any other modified configuration of immunoglobulin molecules comprising an antigen recognition site. Antibodies include any class of antibody, such as IgG, IgA, or IgM (or subclasses thereof), and antibodies are not required to be of any particular class. Immunoglobulins can be classified into different classes according to the antibody amino acid sequence of the heavy chain constant region of the immunoglobulin. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are designated α, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., CD 70). The antigen binding function of an antibody may be performed by a fragment of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab; fab'; fab'; f (ab')₂(ii) a An Fd fragment consisting of the VH and CH1 domains; from antibodies(iii) the Fv fragment consisting of the VL and VH domains of the single arm; single domain antibody (dAb) fragments (Ward et al, Nature 341:544-546,1989) and isolated Complementarity Determining Regions (CDRs).

Antibodies or polypeptides that "preferentially bind" or "specifically bind" (used interchangeably herein) to a target (e.g., CD70 protein) are terms well known in the art, and methods of determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, for a longer period of time, and/or with greater affinity with a particular cell or substance than with a replacement cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds to the target with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a CD70 epitope is one that binds to that epitope with greater affinity, avidity, more readily, and/or with greater duration than to other CD70 epitopes or non-CD 70 epitopes. It is also understood that, by reading this definition, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although may include) exclusive binding. Typically, but not necessarily, reference to binding means preferential binding.

The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of four Framework Regions (FRs) connected by three Complementarity Determining Regions (CDRs), also referred to as hypervariable regions. The CDRs in each chain are held tightly together by the FRs, and the CDRs of the other chain contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on sequence differences across species (i.e., Kabat et al, Sequences of Proteins of Immunological Interest, (5 th edition, 1991, National Institutes of Health, Bethesda MD)); (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et Al, 1997, J.Molec.biol.273: 927-948). As used herein, a CDR may refer to a CDR defined by either method or by a combination of both methods.

The "CDRs" of a variable domain are amino acid residues within the variable region identified according to the definitions and/or conformational definitions of Kabat, Chothia, both Kabat and Chothia, AbM, contact, or any CDR determination method well known in the art. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al, 1992, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, NIH, Washington D.C. The position of the CDRs can also be identified as the structural loop structure originally described by Chothia et al and others. See, e.g., Chothia et al, Nature 342:877-883, 1989. Other CDR identification methods include "AbM definition" (which is a trade-off between Kabat and Chothia, is the use of Oxford Molecular AbM antibody modeling software (now called as the "AbM antibody model"))

) Derivatized), or "contact definition" of CDRs based on observed antigen contact, as described by maccall et al, j.mol.biol.,262:732-745, 1996. In another approach, referred to herein as "conformational definition" of a CDR, the position of the CDR can be identified as a residue that contributes enthalpically to antigen binding. See, for example, Makabe et al, Journal of Biological Chemistry,283: 1156-. Still other CDR boundary definitions may not strictly follow one of the above methods, but will still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened according to the following predicted or experimental findings: neither a particular residue or group of residues, nor even the entire CDR, significantly affects antigen binding. As used herein, a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein may employ CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, a CDR can be defined according to any of Kabat, Chothia, extension, AbM, contact, and/or conformational definitions.

As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies have high specificity (for a single antigenic site). Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, in contrast to polyclonal antibody preparations which typically comprise different antibodies directed against different determinants (epitopes). The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use according to the invention can be prepared by the hybridoma method first described by Kohler and Milstein, Nature 256:495,1975, or can be prepared by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated using techniques such as those described in McCafferty et al, Nature 348:552-554, 1990.

As used herein, "humanized" antibody refers to a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab ', F (ab')₂Or other antigen binding subsequence of an antibody). Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced with residues from a CDR of a non-human species (such as mouse, rat or rabbit) (donor antibody) having the desired specificity, affinity and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not present in either the recipient antibody or the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the CDR regionsAll FR regions are herein those of the human immunoglobulin consensus sequence. The humanized antibody will optimally also comprise at least a portion of an immunoglobulin (typically a human immunoglobulin) constant region or constant domain (Fc). Preferred are antibodies having a modified Fc region as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 or CDR H3) that have been altered relative to the original antibody, also referred to as "derived from" one or more CDRs of the original antibody.

As used herein, "human antibody" means an antibody having an amino acid sequence corresponding to an antibody produced by a human and/or that has been prepared using any of the techniques for preparing human antibodies known to those skilled in the art or disclosed herein. This definition of human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses human antibodies (Vaughan et al, Nature Biotechnology,14: 309-. Human antibodies can also be prepared by immunization of animals into which human immunoglobulin loci have been introduced by transgenes in place of endogenous loci, e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated. This method is described in U.S. patent nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; and 5,661,016. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies to the target antigen (such B lymphocytes can be recovered from an individual or from single cell clones of cDNA, or can have been immunized in vitro). See, e.g., Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77, 1985; boerner et al, J.Immunol.147 (1):86-95,1991; and U.S. Pat. No. 5,750,373.

The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to a chain of amino acids of any length. For example, the chain may be relatively short (e.g., 10-100 amino acids) or longer. The chain may be straight or branched, it may comprise modified amino acids, and/or it may be interrupted by non-amino acids. The term also encompasses amino acid chains that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling component. Also included within this definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides may be present as single chains or associated chains.

A "monovalent antibody" (e.g., IgG or Fab) comprises one antigen binding site per molecule. In some cases, a monovalent antibody may have more than one antigen binding site, but the binding sites are from different antigens.

A "monospecific antibody" (e.g., IgG) comprises two identical antigen binding sites per molecule, such that both binding sites bind the same epitope on the antigen. Thus, they compete with each other for binding to one antigenic molecule. Most antibodies found in nature are monospecific. In some cases, the monospecific antibody may also be a monovalent antibody (e.g., Fab).

A "bivalent antibody" (e.g., IgG) comprises two antigen binding sites per molecule. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies may be bispecific.

"bispecific" or "dual specificity" is a hybrid antibody having two distinct antigen-binding sites. The two antigen binding sites of a bispecific antibody bind to two different epitopes, which may be located on the same or different protein targets.

A "bifunctional" antibody is an antibody that has the same antigen binding site (i.e., the same amino acid sequence) in both arms, but can recognize two different antigens per binding site.

A "heteromultimer", "heteromultimeric complex" or "heteromultimeric polypeptide" is a molecule comprising at least a first polypeptide and a second polypeptide, wherein the amino acid sequence of the second polypeptide differs from the first polypeptide by at least one amino acid residue. The heteromultimer can comprise a "heterodimer" formed by the first polypeptide and the second polypeptide, or can form a higher order tertiary structure in which polypeptides other than the first polypeptide and the second polypeptide are also present.

A "heterodimer", "heterodimeric protein", "heterodimeric complex", or "heteromultimeric polypeptide" is a molecule comprising a first polypeptide and a second polypeptide, wherein the amino acid sequence of the second polypeptide differs from the amino acid sequence of the first polypeptide by at least one amino acid residue.

As used herein, "hinge region", "hinge sequence" and variants thereof include those known in the art, as described, for example, in Janeway et al, immunology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4 th edition, 1999); bloom et al, Protein Science (1997),6: 407-; humphreys et al, J.Immunol.methods (1997),209: 193-202.

As used herein, "immunoglobulin-like hinge region," "immunoglobulin-like hinge sequence," and variants thereof refer to the hinge region and hinge sequence of an immunoglobulin-like or antibody-like molecule (e.g., immunoadhesin). In some embodiments, the immunoglobulin-like hinge region may be derived or derived from any IgG1, IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD, or IgM, including chimeric versions thereof, such as a chimeric IgG1/2 hinge region.

As used herein, the term "immune effector cell" or "effector cell" refers to a cell within a natural cell pool in the human immune system that can be activated to affect the viability of a target cell. Viability of a target cell may include the ability of the cell to survive, proliferate, and/or interact with other cells.

Antibodies of the invention can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or combinations of these techniques or other techniques readily known in the art (see, e.g., Jayasena, S.D., Clin.Chem.,45: 1628-.

As known in the art, "polynucleotide" or "nucleic acid" used interchangeably herein refers to a chain of nucleotides of any length and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into the strand by DNA or RNA polymerase. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. Modification of the nucleotide structure, if present, may be performed before or after strand assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", the replacement of one or more of the naturally occurring nucleotides by an analog, internucleotide modifications (e.g., those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of one or more polynucleotides. In addition, they are usually present in sugarsAny of the hydroxyl groups in (a) may be replaced, for example, by a phosphonic acid group, a phosphate group protected by a standard protecting group, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to a solid support. The 5 'and 3' terminal OH groups may be phosphorylated or partially substituted with an amine or an organic capping group having 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized as standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose commonly known in the art, including, for example, 2 '-O-methyl ribose, 2' -O-allyl ribose, 2 '-fluoro ribose or 2' -azidoribose, carbocyclic sugar analogs, alpha-or beta-anomeric sugars, epimeric sugars (such as arabinose, xylose, or lyxose), pyranoses, furanoses, sedoheptulose, acyclic analogs, and alkali-free nucleoside analogs (such as methyl ribonucleosides). One or more phosphodiester linkages may be substituted with an alternative linking group. Such alternative linking groups include, but are not limited to, phosphate by P (O) S ("thiophosphate"), P (S) S ("dithiophosphate"), (O) NR₂("amide"), P (O) R, P (O) OR', CO OR CH₂("methylal") alternative embodiment, wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 carbons) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl (araldyl). It is not required that all linkages in a polynucleotide be identical. The above description applies to all polynucleotides mentioned herein, including RNA and DNA.

As known in the art, a "constant region" of an antibody refers to either the constant region of an antibody light chain or the constant region of an antibody heavy chain, alone or in combination.

As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, yet more preferably at least 98% pure, and most preferably at least 99% pure.

"host cell" includes a single cell or cell culture which may be or has been the recipient of one or more vectors for incorporation of a polynucleotide insert. Host cells include progeny of a single host cell, and the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with one or more polynucleotides of the invention.

The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, as known in the art. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of immunoglobulin heavy chains may vary, the human IgG heavy chain Fc region is generally defined as the stretch of sequence from the amino acid residue at position Cys226 or from position Pro230 to their carboxy terminus. The numbering of residues in the Fc region is that of the EU index as described in Kabat. Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin typically comprises two constant domains, CH2 and CH 3.

As used in the art, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, preferred fcrs are those which bind IgG antibodies (gamma receptors) and include receptors of the Fc γ RI, Fc γ RII and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcR in ravatch and Kinet, ann.rev.immunol.,9: 457-; capel et al, immunolmethods, 4:25-34, 1994; and de Haas et al, J.Lab.Clin.Med.,126: 330-. "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.,117:587,1976; and Kim et al, J.Immunol.,24:249,1994).

As used herein, the term "competes" with respect to an antibody means that the first antibody, or antigen-binding fragment (or portion) thereof, binds to an epitope in a manner sufficiently similar to the binding of the second antibody, or antigen-binding portion thereof, such that the binding of the first antibody to its cognate epitope in the presence of the second antibody is detectably reduced as compared to the binding of the first antibody in the absence of the second antibody. An alternative where the binding of the second antibody to its epitope in the presence of the first antibody is also detectably reduced may be, but is not required to be, such. That is, the first antibody may inhibit the binding of the second antibody to its epitope, while the second antibody does not inhibit the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of another antibody to its cognate epitope or ligand (whether to the same, greater, or lesser extent), the antibodies are said to "cross-compete" with each other for binding to their respective epitope or epitopes. The present invention encompasses both competitive and cross-competitive antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), the skilled artisan will appreciate, in light of the teachings provided herein: such competitive and/or cross-competitive antibodies are encompassed and useful in the methods disclosed herein.

A "functional Fc region" has at least one effector function of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions typically require an Fc region in combination with a binding domain (e.g., an antibody variable domain), and can be evaluated using various assays known in the art for evaluating such antibody effector functions.

A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution, for example from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the Fc region of the native sequence or in the Fc region of the parent polypeptide as compared to the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc regions herein preferably have at least about 80% sequence identity with the native sequence Fc region and/or the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity therewith, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.

The term "effector function" refers to a biological activity attributable to the Fc region of an antibody. Examples of antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, complement-dependent cytotoxicity (CDC), phagocytosis, C1q binding, and down-regulation of cell surface receptors (e.g., B cell receptors; BCR). See, for example, U.S. Pat. No. 6,737,056. Such effector functions typically require an Fc region in combination with a binding domain (e.g., an antibody variable domain), and can be evaluated using various assays known in the art for evaluating such antibody effector functions. Exemplary measurements of effector function are performed by Fc γ 3 and/or C1q binding.

As used herein, "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize antibody bound on a target cell and subsequently cause lysis of the target cell. ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, such as those described in U.S. patent No. 5,500,362 or 5,821,337. Effector cells for use in such assays include Peripheral Blood Mononuclear Cells (PBMC) and NK cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed by Clynes et al, 1998, PNAS (USA),95: 652-.

"complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) that is complexed to a cognate antigen. To assess complement activation, CDC assays can be performed, for example, as described by Gazzano-Santoro et al, J.Immunol.methods,202:163 (1996).

As used herein, "treatment" is a method for obtaining a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing proliferation of (or destroying) neoplastic or cancerous cells, inhibiting metastasis of neoplastic cells, reducing or reducing the size of a tumor that expresses CD70, ameliorating a CD 70-associated disease (e.g., cancer), alleviating a symptom resulting from a CD 70-associated disease (e.g., cancer), increasing the quality of life of a patient having a CD 70-associated disease (e.g., cancer), reducing the dose of other drugs required to treat a CD 70-associated disease (e.g., cancer), delaying the progression of a CD 70-associated disease (e.g., cancer), curing a CD 70-associated disease (e.g., cancer), and/or extending the survival of a patient having a CD 70-associated disease (e.g., cancer).

By "improving" is meant a reduction or improvement in one or more symptoms as compared to not administering the CD70 antibody (monospecific or bispecific). "improving" also includes shortening or reducing the duration of symptoms.

As used herein, an "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological, and/or behavioral symptoms of the disease, complications of the disease, and intermediate pathological phenotypes that arise during the development of the disease. For therapeutic use, beneficial or desired results include the following clinical results: such as reducing the incidence of or ameliorating one or more symptoms of various CD 70-associated diseases or disorders (such as, for example, multiple myeloma), reducing the dosage of other drugs required to treat the disease, and/or delaying the progression of CD 70-associated disease in a patient. An effective dose may be administered in one or more administrations. For the purposes of the present invention, an effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, prophylactic or therapeutic treatment. As understood in the clinical context, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective dose" can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to be administered in an effective amount if it can achieve, or has achieved, the desired result in combination with one or more other agents.

An "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, primates, horses, dogs, cats, mice, and rats.

As used herein, "vector" means a construct capable of delivery and preferably expression of one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (such as producer cells).

As used herein, "expression control sequence" means a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence may be a promoter (such as a constitutive or inducible promoter) or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active component, allows the component to retain biological activity and not react with the immune system of a subject. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and any of various types of wetting agents. Preferred diluents for aerosol or parenteral administration are Phosphate Buffered Saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition, A.Gennaro eds., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21 st edition, Mack Publishing, 2005).

As used herein, the term "acyl donor glutamine-containing tag" or "glutamine tag" refers to a polypeptide or protein that contains one or more Gln residues that serve as amine acceptors for transglutaminase. See, for example, WO2012059882 and WO 2015015448.

As used herein, the term "k_on"or" k_a"refers to the rate constant for the association of an antibody with an antigen. Specifically, the rate constant (k)_on/k_aAnd k_off/k_d) And equilibrium dissociation constants were measured using a full antibody (i.e., bivalent) and a monomeric CD70 protein (e.g., a histidine-tagged CD70 fusion protein).

As used herein, the term "k_off"or" k_d"refers to the rate constant at which an antibody dissociates from an antibody/antigen complex.

As used herein, the term "K_D"refers to the equilibrium dissociation constant of an antibody-antigen interaction.

Reference herein to "about" a value or parameter includes (and describes) embodiments that relate to that value or parameter per se. For example, a description referring to "about X" includes a description of "X". Numerical ranges include the numbers defining the range. In general, the term "about" refers to a specified value of a variable and all values of the variable that are within experimental error of the specified value (e.g., within 95% confidence interval of the mean) or within 10% of the specified value (whichever is larger). Where the term "about" is used in the context of a period of time (year, month, week, day, etc.), the term "about" means that the period of time is plus or minus the amount of the next subordinate period of time (e.g., about 1 year means 11-13 months; about 6 months means 6 months plus or minus 1 week; about 1 week means 6-8 days; etc.), or within 10% of the stated value, whichever is greater.

It should be understood that wherever the language "comprising" is used herein to describe an embodiment, other similar embodiments described as "consisting of … …" and/or "consisting essentially of … …" are additionally provided.

Where aspects or embodiments of the invention are described in terms of the markush group or other alternative group, the invention encompasses not only the entire group listed as a whole, but also each member of the group and all possible sub-groups of the total group individually, as well as the total group in the absence of one or more group members. The present invention also contemplates explicitly excluding one or more of any group members from the claimed invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

Although methods and materials similar or equivalent to those described herein can additionally be used in the practice or testing of the present invention, exemplary methods and materials are described herein. These materials, methods, and examples are illustrative only and not intended to be limiting.

CD70 antibody and preparation method thereof

The invention provides an antibody that binds to CD70[ e.g., human CD70 (e.g., accession No.: NP _004110 or SEQ ID No.: 235) ] characterized by any one or more of the following features: (a) treating, preventing, ameliorating one or more symptoms of a disorder associated with malignant cells that express CD70 (e.g., cancer such as AML) in a subject; (b) inhibiting tumor growth or progression in a subject (a subject having a malignancy that expresses CD 70); (c) inhibiting metastasis of cancer (malignant) cells expressing CD70 in a subject (a subject with one or more malignancies expressing CD 70); (d) inducing regression (e.g., long-term regression) of CD 70-expressing tumors; (e) exert cytotoxic activity in malignant cells expressing CD 70; (f) block the interaction of CD70 with other yet unidentified factors; and/or (g) inducing a bystander effect that kills nearby non-CD 70 expressing malignant cells or inhibits the growth of those cells.

In one aspect, there is provided an isolated antibody that specifically binds to CD70, wherein the antibody comprises (a) a heavy chain Variable (VH) region comprising: (i) VH complementarity determining region 1(CDR1) comprising 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 405, 363, 364, 368, 369, 370, 375, 392, 376, 380, 381, 382, 386, 398, 412, 423, 410, 418, 410, 416, 374, 410, 374, 87, 382, 428. 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459, or 460; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 449, 450, 455, 456, 461, or 462; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457 or 463; and/or a light chain Variable (VL) region comprising: (i) a VL CDR1 comprising the sequence set forth in SEQ ID NO 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, or 527; (ii) a VL CDR2 comprising a sequence set forth in SEQ ID NO 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, or 528; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526 or 529.

In another aspect, there is provided an isolated antibody that specifically binds to CD70, wherein the antibody comprises: a VH region comprising a VH CDR1, VH CDR2 and VH CDR3 of the VH sequence set forth in SEQ ID NOs 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329 or 331; and/or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NOs 1,3, 5,7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, or 330.

In some embodiments, antibodies having any one of the partial light chain sequences listed in table 1 and/or the partial heavy chain sequences listed in table 1 are provided. In table 1, the underlined sequences are CDR sequences according to Kabat, and the bold sequences are sequences according to Chothia.

TABLE 1

Also provided herein are CDR portions of the antigen binding domains of antibodies directed to CD70 (including Chothia, Kabat CDRs, and CDR contact regions). Determination of CDR regions is well within the skill of the art. It is understood that in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as "combination CR" or "extended CDRs"). In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDR is a Chothia CDR. In other words, in embodiments having more than one CDR, the CDR can be any of Kabat, Chothia, a combination CDR, or a combination thereof. Table 2 provides examples of CDR sequences provided herein.

TABLE 2

In some embodiments, the invention provides antibodies that bind to CD70 and compete with antibodies as described herein, including 31H1, 63B2, 40E3, 42C3, 45F11, 64F 11, 72C 11, 2F 11, 4F11, 10H 11, 17G 11, 65E11, P02B 11, P07D 11, P08a 11, P08E 11, P08F 11, P08G 11, P12B 11, P12F 11, P12G 11, P13F 11, P15D 11, P16C 11, P10 a 11, 10E 11, 11a 11, 11C 11, 11D 11, 11E 11, 12a 11, 12C 11, 12D 11, 11D 11, 369F 369, 11, 369, or 369.

In some embodiments, the invention also provides CDR portions of antibodies directed to the CD70 antibody based on the CDR contact regions. A CDR contact region is a region of an antibody that confers specificity for an antigen to the antibody. Typically, the CDR contact region includes residue positions in the CDR and Vernier zones that are constrained so as to maintain an appropriate loop structure for the antibody to bind to a particular antigen. See, e.g., Makabe et al, J.biol.chem.,283: 1156-. Determination of the CDR contact area is well within the skill of the art.

CD70 antibodies as described hereinBinding affinity (K) of a body to CD70, such as human CD70 (e.g., (SEQ ID NO:278)))_D) Can be from about 0.001 to about 5000 nM. In some embodiments, the binding affinity is about 5000nM, 4500nM, 4000nM, 3500nM, 3000nM, 2500nM, 2000nM, 1789nM, 1583nM, 1540nM, 1500nM, 1490nM, 1064nM, 1000nM, 933nM, 894nM, 750nM, 705nM, 678nM, 532nM, 500nM, 494nM, 400nM, 349nM, 340nM, 353nM, 300nM, 250nM, 244nM, 231nM, 225nM, 207nM, 200nM, 186nM, 172nM, 136nM, 113nM, 104nM, 101nM, 100nM, 90nM, 83nM, 79nM, 74nM, 54nM, 50nM, 45nM, 42nM, 40nM, 35nM, 32nM, 30nM, 25nM, 24nM, 22nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 12nM, 10nM, 9nM, 8nM, 7.5nM, 7nM, 6.5nM, 6nM, 5.5nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.5nM, 0.3nM, 0.1nM, 0.01nM, or 0.001 nM. In some embodiments, the binding affinity is less than any one of about 5000nM, 4000nM, 3000nM, 2000nM, 1000nM, 900nM, 800nM, 250nM, 200nM, 100nM, 50nM, 30nM, 20nM, 10nM, 7.5nM, 7nM, 6.5nM, 6nM, 5nM, 4.5nM, 4nM, 3.5nM, 3nM, 2.5nM, 2nM, 1.5nM, 1nM, or 0.5 nM.

Bispecific antibodies, i.e., monoclonal antibodies having binding specificity for at least two different antigens, can be prepared using the antibodies disclosed herein. Methods for making bispecific antibodies are known in the art (see, e.g., Suresh et al, Methods in Enzymology 121:210,1986). Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy-light chain pairs, the two heavy chains having different specificities (Millstein and Cuello, Nature 305,537-539, 1983). Thus, in one aspect, a bispecific antibody is provided, wherein the bispecific antibody is a full length human antibody comprising a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen (e.g., CD70), and a second antibody variable domain of the bispecific antibody that is capable of recruiting the activity of a human immune effector cell by specifically binding to an effector antigen localized on the human immune effector cell.

The human immune effector cell may be any of a variety of immune effector cells known in the art. For example, the immune effector cell can be a member of the human lymphocyte lineage, including but not limited to T cells (e.g., cytotoxic T cells), B cells, and Natural Killer (NK) cells. Immune effector cells may also be, for example, but not limited to, members of the human myeloid lineage, including, but not limited to, monocytes, neutrophils, and dendritic cells. Such immune effector cells may have cytotoxic or apoptotic effects on target cells, or other desired effects upon activation through binding of effector antigens.

An effector antigen is an antigen (e.g., a protein or polypeptide) that is expressed on human immune effector cells. Examples of effector antigens that can be bound by heterodimeric proteins (e.g., heterodimeric antibodies or bispecific antibodies) include, but are not limited to, human CD3 (or CD3 (cluster of differentiation) complex), CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD 89.

The target cell may be a human native or foreign cell. In a native target cell, the cell may have been transformed into a malignant cell or be pathologically modified (e.g., a native target cell infected with a virus, plasmodium, or bacterium). In exogenous target cells, the cells are infiltrating pathogens such as bacteria, plasmodium or viruses.

In a disease condition (e.g., an inflammatory disease, a proliferative disease (e.g., cancer), an immune disorder, a neurological disease, a neurodegenerative disease, an autoimmune disease, an infectious disease (e.g., a viral infection or a parasitic infection), an allergic reaction, a graft-versus-host disease, or a host-versus-graft disease), a target antigen is expressed on a target cell. The target antigen is not an effector antigen. In some embodiments, the target antigen is CD 70.

In some embodiments, a bispecific antibody is provided, wherein the bispecific antibody is a full length antibody comprising a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen and a second antibody variable domain of the bispecific antibody capable of recruiting the activity of a human immune effector cell by specifically binding to an effector antigen localized on the human immune effector cell, wherein the first antibody variable domain comprises a heavy chain Variable (VH) region comprising SEQ ID NOs 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 2, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 293, 295, 297, 299, 301, 303, 305, 307. 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329 or 331, VH CDR1, VH CDR2 and VH CDR3 of the VH sequence; and/or a light chain Variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NOs 1,3, 5,7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, or 330.

In some embodiments, there is provided a bispecific antibody, wherein the bispecific antibody is a full length antibody comprising a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen, and a second antibody variable domain of the bispecific antibody capable of recruiting the activity of a human immune effector cell by specifically binding to an effector antigen localized on the human immune effector cell, wherein the first antibody variable domain comprises (a) a heavy chain Variable (VH) region comprising: (i) VH complementarity determining region 1(CDR1) comprising SEQ ID NOs 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 406, 368, 369, 370, 368, 375, 392, 380, 376, 382, 398, 393, 410, 416, 220, 2, 382, 393, 2, 393, 410, 393, and 393, 410, 422. 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459, or 460; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 449, 450, 455, 456, 461, or 462; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457 or 463; and/or a light chain Variable (VL) region comprising: (i) a VL CDR1 comprising the sequence set forth in SEQ ID NO 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, or 527; (ii) a VL CDR2 comprising a sequence set forth in SEQ ID NO 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, or 528; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526 or 529.

In some embodiments, the second antibody variable domain comprises a heavy chain Variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 266; and/or a light chain variable region (VL) comprising the VL CDR1, VL CDR2 and VL CDR3 of the VL sequence shown in SEQ ID NO: 265.

In some embodiments, the second antibody variable domain comprises (a) a heavy chain Variable (VH) region comprising: (i) VH complementarity determining region 1(CDR1) comprising the sequence shown in SEQ ID NOs: 267, 268, or 269; (ii) VH CDR2 comprising the sequence shown in SEQ ID NO 270 or 271; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 272; and/or (b) a light chain Variable (VL) region comprising: (i) VL CDR1 comprising the sequence shown in SEQ ID NO: 273; (ii) VL CDR2 comprising the sequence shown in SEQ ID NO. 274; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 275.

Table 3 shows specific amino acid and nucleic acid sequences of the variable domains of the second antibody, which sequences are specific for CD 3. In table 3, the underlined sequences are CDR sequences according to Kabat, and the bold sequences are sequences according to Chothia.

TABLE 3

Table 4 shows an example of the CDR sequences of the variable domains of the second antibody, which sequences are specific for CD 3.

TABLE 4

In some embodiments, bispecific antibodies provided herein contain a CD 3-specific variable domain having an anti-CD 3 sequence as provided in U.S. publication No. 20160297885, which is hereby incorporated by reference for all purposes.

According to one method of making bispecific antibodies, antibody variable domains having the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant region sequences. The fusion is preferably to an immunoglobulin heavy chain constant region comprising at least a portion of the hinge, CH2, and CH3 regions. Preferably, a first heavy chain constant region (CH1) is present in at least one of the fusions, said first heavy chain constant region comprising the site required for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides in embodiments great flexibility in adjusting the mutual proportions of the three polypeptide fragments, when the optimal yield is obtained for the three polypeptide chains used in the unequal ratio construction. However, when expression of equal proportions of at least two polypeptide chains yields high yields or when the proportions are of no particular significance, the coding sequences for two or all three polypeptide chains can be inserted into one expression vector.

In another approach, a bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has immunoglobulin light chains in only half of the bispecific molecule, facilitating the separation of the desired bispecific compound from the undesired immunoglobulin chain combinations. Such a process is described in PCT publication No. WO 94/04690.

In another approach, bispecific antibodies consist of amino acid modifications in the first hinge region in one arm and substituted/substituted amino acids in the first hinge region (of opposite charge to the corresponding amino acids in the second hinge region in the other arm). Such a process is described in international patent application No. PCT/US2011/036419(WO 2011/143545).

In another approach, formation of a desired heteromultimeric or heterodimeric protein (e.g., a bispecific antibody) is enhanced by altering or engineering the interface between a first immunoglobulin-like Fc region and a second immunoglobulin-like Fc region (e.g., a hinge region and/or a CH3 region). In this approach, the bispecific antibody may consist of a CH3 region, wherein the CH3 region comprises a first CH3 polypeptide and a second CH3 polypeptide that interact together to form a CH3 interface, wherein one or more amino acids within the CH3 interface destabilize homodimer formation and are electrostatically disfavored for homodimer formation. Such a process is described in international patent application No. PCT/US2011/036419(WO 2011/143545).

In another approach, bispecific antibodies can be generated using a glutamine-containing peptide tag engineered as an antibody to an epitope in one arm (e.g., CD70) and another peptide tag of a second antibody engineered as a second antibody to a second epitope in the other arm in the presence of transglutaminase (e.g., a Lys-containing peptide tag or reactive endogenous Lys). Such a process is described in international patent application No. PCT/IB2011/054899(WO 2012/059882).

In some embodiments, a heterodimeric protein (e.g., a bispecific antibody) as described herein comprises a full-length human antibody, wherein a first antibody variable domain of the bispecific antibody specifically binds to a target antigen (e.g., CD70), and comprises a second antibody variable domain of the bispecific antibody capable of recruiting the activity of a human immune effector cell by specifically binding to an effector antigen (e.g., CD3) located on the human immune effector cell, wherein the first and second antibody variable domains of the heterodimeric protein comprise amino acid modifications at positions 223, 225, and 228 in the hinge region (e.g., (C223E or C223R), (E225E or E225R), and (P228E or P228R)) and amino acid modifications at positions 409 or 368 in the CH3 region (SEQ ID NO:279) of human IgG2 (e.g., K409R or L368E (EU numbering scheme)).

In some embodiments, the first and second antibody variable domains of the heterodimeric protein comprise amino acid modifications at positions 221 and 228 in the hinge region (e.g., (D221R or D221E) and (P228R or P228E)) and amino acid modifications at positions 409 or 368 in the CH3 region of human IgG1 (SEQ ID NO:280) (e.g., K409R or L368E (EU numbering scheme)).

In some embodiments, the first antibody variable domain and the second antibody variable domain of the heterodimeric protein comprise an amino acid modification at position 228 in the hinge region (e.g., (P228E or P228R)) and an amino acid modification at position 409 or 368 in the CH3 region (SEQ ID NO:281) of human IgG4 (e.g., R409 or L368E (EU numbering scheme)).

Antibodies useful in the invention can encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab ', F (ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion (e.g., a domain antibody), humanized antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of the desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody may be murine, rat, human, or any other source of antibodies (including chimeric or humanized antibodies).

In some embodiments, a CD70 monospecific antibody or a CD70 bispecific antibody (e.g., CD70-CD3) as described herein is a monoclonal antibody. For example, the CD70 monospecific antibody is a human monoclonal antibody. In another example, the CD70 arm of the CD70-CD3 bispecific antibody is a human monoclonal antibody and the CD3 arm of the CD70-CD3 bispecific antibody is a humanized monoclonal antibody.

In some embodiments, the antibody comprises a modified constant region, such as, but not limited to, a constant region with increased potential to elicit an immune response. For example, the constant region can be modified to have increased affinity for an Fc γ receptor (e.g., Fc γ RI, Fc γ RIIA, or Fc γ III).

In some embodiments, the antibody comprises a modified constant region, such as a constant region that is immunologically inert (i.e., has reduced potential to elicit an immune response). In some embodiments, the constant region is as defined in eur.j.immunol.,29:2613-2624, 1999; PCT application No. PCT/GB 99/01441; and/or as described in UK patent application No. 98099518. The Fc may be human IgG1, human IgG2, human IgG3, or human IgG 4. The Fc may be human IgG2(IgG2 Δ a) containing the mutations a330P331 to S330S331, with the amino acid residues numbered with reference to the wild-type IgG2 sequence. Eur.J.Immunol.,29:2613-2624, 1999. In some embodiments, the antibody comprises an IgG comprising the following mutations₄The constant region of (Armour et al, Molecular Immunology 40585-: E233F234L235 to P233V234a235(IgG4 Δ c), wherein numbering is performed with reference to wild-type IgG 4. In yet another embodiment, Fc is human IgG4E233F234L235 to P233V234a235 with deletion G236(IgG4 Δ b). In another embodiment, the Fc is any human IgG4 Fc (IgG4, IgG4 Δ b, or IgG4 Δ c) containing the hinge stabilizing mutations S228 to P228 (aalbese et al, Immunology 105,9-19,2002). In another embodiment, the Fc may be a non-glycosylated Fc.

In some embodiments, the constant region is non-glycosylated by mutation of an oligosaccharide attachment residue (such as Asn297) and/or a residue flanking the constant region that is part of the glycosylation recognition sequence. In some embodiments, the constant region is non-glycosylated by an enzyme directed to N-linked glycosylation. The constant region may be non-glycosylated either enzymatically for N-linked glycosylation or by expression in a host cell deficient in glycosylation.

In some embodiments, the constant region has a modified constant region that removes or reduces Fc γ receptor binding. For example, Fc may be human IgG2 containing the mutation D265, wherein the amino acid residues are numbered with reference to the wild-type IgG2 sequence (SEQ ID NO: 279). Thus, in some embodiments, the constant region has the modified constant region of the sequence set forth in SEQ ID NO 282: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCRVRCPRCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK are provided. And the nucleic acid encoding the sequence shown by SEQ ID NO 282 is shown as SEQ ID NO 283.

In some embodiments, the constant region has a modified constant region having the sequence shown in SEQ ID NO 284:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK are provided. And the nucleic acid encoding the sequence shown in SEQ ID NO 284 is shown in SEQ ID NO 285.

The amino acids of the human kappa constant region are shown in SEQ ID NO: 286: GTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC are provided. And the nucleic acid encoding the sequence of SEQ ID NO 286 is shown as SEQ ID NO 287.

One method of determining the binding affinity of an antibody to CD70 is by measuring the binding affinity of a bivalent antibody to monomeric CD70 protein. The affinity of the CD70 antibody can be determined by surface plasmon resonance (Biacore)^TM3000^TMSurface Plasmon Resonance (SPR) system, Biacore^TMINC, Piscataway NJ) using HBS-EP running buffer (0.01M HEPES pH 7.4, 0.15NaCl, 3mM EDTA, 0.005% v/v surfactant P20) with pre-immobilized anti-mouse Fc or anti-human Fc. Monomeric 8 histidine-tagged human CD70 extracellular domain (SEQ ID NO:530) can be diluted into HBS-EP buffer to a concentration of less than 0.5. mu.g/mL and injected into each individual chip channel using variable contact times to achieve two antigen density ranges, i.e., 50-200 reactions for detailed kinetic studiesUnits (RU), or 800-1,000RU for screening assays. Regeneration studies showed that 25mM NaOH dissolved in 25% v/v ethanol was effective in removing bound CD70 protein while maintaining the activity of CD70 antibody on the chip over 200 injections. Typically, serial dilutions (spanning concentrations of 0.1-10 times estimated K) will be made_D) The purified 8 histidine-tagged CD70 sample (SEQ ID NO:530) of (1) was injected at 100L/min for 1 minute and allowed to dissociate for a period of up to 2 hours. The concentration of CD70 protein was determined by absorbance at 280nm based on the sequence-specific extinction coefficient of the 8 histidine-tagged CD70 protein (SEQ ID NO: 530). Kinetic association rates (k. times. Enzymology 6.99-110) were obtained simultaneously by global fitting of data to a 1:1 Langmuir (Langmuir) binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994) using the BIAevaluation program_onOr k_a) And dissociation rate (k)_offOr k_d). Equilibrium dissociation constant (K)_D) The value is calculated by the formula k_off/k_on. This protocol is applicable to determine the binding affinity of an antibody to any monomeric CD70, including human CD70, another mammalian CD70 (such as mouse CD70, rat CD70, or primate CD70), and a different form of CD70 (e.g., glycosylated CD 70). The binding affinity of an antibody is typically measured at 25 ℃, but can also be measured at 37 ℃.

Antibodies as described herein can be prepared by any method known in the art. For the generation of hybridoma cell lines, the route and schedule of immunization of the host animal is generally consistent with established and conventional antibody stimulation and production techniques, as further described herein. General techniques for producing human and mouse antibodies are known in the art and/or described herein.

It is contemplated that any mammalian subject, including humans or cells producing antibodies therefrom, can be manipulated to serve as a basis for mammalian (including human and hybridoma cell lines) production. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of an immunogen (including an immunogen as described herein).

Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic hybridization technique of Kohler, B, and Milstein, C., Nature 256:495-497,1975 or the technique of Buck, D.W., et al, In Vitro,18:377-381, 1982. Useful myeloma Cell lines (including but not limited to X63-Ag8.653 and those from Salk Institute, Cell Distribution Center, San Diego, Calif., USA) can be used for hybridization. Generally, the technique involves myeloma and lymphocyte fusion using a fusing agent (such as polyethylene glycol) or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parental cells. Any of the media described herein, with or without serum supplementation, can be used to culture monoclonal antibody-secreting hybridomas. As another alternative to cell fusion techniques, EBV immortalized B cells can be used to produce the monoclonal antibodies of the subject invention. If desired, the hybridomas are expanded and subcloned, and the supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that can be used as a source of antibodies encompass all derivatives, progeny cells, of the parent hybridoma that produce a monoclonal antibody specific for CD70 or portions thereof.

Hybridomas producing such antibodies can be grown in vitro or in vivo using known procedures. If desired, monoclonal antibodies can be isolated from the culture medium or body fluids by conventional immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. If present, undesired activity can be removed, for example, by running the preparation on an adsorbent (made of the immunogen attached to a solid phase) and eluting or releasing the desired antibody from the immunogen. By expressing human CD70, human CD70 protein or containing a protein conjugated to a protein immunogenic in the species to be immunized (e.g., openpore snail hemocyanin, serum albumin, bovine thyroglobulin or using bifunctionalOr a derivatizing agent such as maleimidobenzoyl sulfosuccinimide ester (conjugated through a cysteine residue), N-hydroxysuccinimide (conjugated through a lysine residue), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N ═ C ═ NR, where R and R¹Are different alkyl groups) to immunize a host animal to produce a population of antibodies (e.g., monoclonal antibodies).

If desired, the antibody of interest (monoclonal or polyclonal) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequences encoding the antibody of interest may be maintained in a vector in a host cell, which may then be expanded and frozen for future use. Production of recombinant monoclonal antibodies in cell culture can be performed by cloning antibody genes from B cells using means known in the art. See, e.g., Tiller et al, j.immunol.methods 329,112,2008; U.S. patent No. 7,314,622.

In one alternative, the polynucleotide sequences may be used for genetic manipulation to "humanize" the antibody or to improve the affinity or other characteristics of the antibody. For example, if the antibody is used in clinical trials and treatments for humans, the constant region can be engineered to more closely resemble a human constant region to avoid an immune response. It may be desirable to genetically manipulate antibody sequences to obtain greater affinity for CD70 and greater efficacy in inhibiting CD 70.

There are four general steps for humanizing monoclonal antibodies. They are: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy chain variable domains, (2) designing a humanized antibody, i.e., determining which antibody framework regions to use in the humanization process, (3) the actual humanization method/technique, and (4) transfection and expression of the humanized antibody. See, for example, U.S. Pat. nos. 4,816,567, 5,807,715, 5,866,692, 6,331,415, 5,530,101, 5,693,761, 5,693,762, 5,585,089, and 6,180,370.

Many "humanized" antibody molecules have been described that comprise an antigen binding site derived from a non-human immunoglobulin, including chimeric antibodies having rodent or modified rodent V regions and their associated CDRs fused to human constant regions. See, for example, Winter et al Nature 349: 293-. Other references describe rodent CDRs grafted onto human supporting Framework (FR) regions prior to fusion with appropriate human antibody constant regions. See, for example, Riechmann et al Nature 332: 323-. Another reference describes rodent CDRs supported by recombinantly engineered rodent framework regions. See, for example, european patent publication No. 0519596. These "humanized" molecules are designed to minimize the undesirable immune response to rodent anti-human antibody molecules, which limits the duration and effectiveness of therapeutic applications of these moieties in human recipients. For example, the antibody constant region can be engineered to be immunologically inert (e.g., not triggering complement lysis). See, e.g., PCT publication Nos. PCT/GB 99/01441; british patent application No. 9809951.8. Other humanization methods of antibodies that may additionally be utilized are described in Daugherty et al, Nucl. acids Res.19:2471-2476,1991, and U.S. Pat. Nos. 6,180,377, 6,054,297, 5,997,867, 5,866,692, 6,210,671 and 6,350,861; and PCT publication No. WO 01/27160.

The general principles discussed above in relation to humanized antibodies are also applicable to customized antibodies that can be used, for example, in dogs, cats, primates, horses and cattle. Furthermore, one or more aspects of the humanized antibodies described herein, such as CDR grafting, framework mutations, and CDR mutations, can be combined.

In one variant, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulins. Designed to produce more desirable (e.g., fully human antibodies) or more robustThe immune-responsive transgenic animals of (a) can also be used to produce humanized or human antibodies. An example of such a technique is Xenomouse from Abgenix, Inc. (Fremont, CA)^TMAnd from Metarex, Inc. (Princeton, NJ)

And TC Mouse^TM。

In one alternative, the antibody may be recombinantly produced and expressed using any method known in the art. In another alternative, the antibody may be recombinantly produced by phage display technology. See, for example, U.S. Pat. nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150; and Winter et al, Annu.Rev.Immunol.12:433-455, 1994. Alternatively, phage display technology (McCafferty et al, Nature 348:552-553,1990) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene libraries from non-immunized donors. According to this technique, antibody V domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phage (such as M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, the result of selection based on the functional properties of the antibody is also the selection of the gene encoding the antibody exhibiting these properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for a review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564, 571, 1993. Several sources of V gene segments are available for phage display. Clackson et al, Nature 352:624-628,1991 isolated a variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleen of immunized mice. The V gene bank from an unimmunized human donor can be constructed essentially according to the techniques described in the following references, and antibodies to a variety of antigens (including self-antigens) can be isolated: mark et al, J.mol.biol.222:581-597,1991, or Griffith et al, EMBO J.12:725-734, 1993. In the natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutations). Some of the introduced changes will confer higher affinity, and B cells displaying high affinity surface immunoglobulins will preferentially replicate and differentiate during subsequent antigen challenge. This natural process can be simulated by employing a technique called "chain shuffling". (Marks et al, Bio/Technol.10:779-783, 1992). In this approach, the affinity of human "primary antibodies" obtained by phage display can be improved by sequential replacement of the heavy and light chain V region genes with naturally occurring variants (libraries) of the V domain genes obtained from non-immunized donors. This technique allows the generation of antibodies and antibody fragments with affinities in the pM-nM range. Strategies for preparing very large phage antibody libraries (also known as "all-source libraries") have been described by Waterhouse et al, Nucl. acids Res.21: 2265. sup. 2266. 1993. Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibodies have similar affinity and specificity as the starting rodent antibody. According to this method (also known as "epitopic imprinting"), heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced with a human V domain gene bank to generate rodent-human chimeras. The result of the selection performed on the antigen is the isolation of human variable regions capable of restoring a functional antigen binding site, i.e. the epitope determines the choice of (imprinting) partner. When this process is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT publication No. WO 93/06213). Unlike traditional humanization of rodent antibodies by CDR grafting, this technique provides fully human antibodies that lack rodent-derived framework or CDR residues.

The antibody may be recombinantly produced by: antibodies and antibody-producing cells are first isolated from a host animal, gene sequences are obtained, and the antibodies are recombinantly expressed in host cells (e.g., CHO cells) using the gene sequences. Another method that may be employed is the expression of antibody sequences in plants (e.g., tobacco) or transgenic milk. Methods for recombinant expression of antibodies in plants or milk have been disclosed. See, e.g., Peeters et al Vaccine 19:2756,2001; lonberg, n. and d.huskzar int.rev.immunol 13:65, 1995; and Pollock et al, J Immunol Methods 231:147, 1999. Methods for making derivatives of antibodies (e.g., humanized, single chain, etc.) are known in the art.

Immunoassays and flow cytometry sorting techniques, such as Fluorescence Activated Cell Sorting (FACS), can also be used to isolate antibodies specific for CD70 or a tumor antigen of interest.

The antibodies as described herein can be conjugated to a variety of different carriers. The carrier may be active and/or inert. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural and modified cellulose, polyacrylamide, agarose, and magnetite. For the purposes of the present invention, the carrier may be soluble or insoluble in nature. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to determine such antibodies using routine experimentation. In some embodiments, the carrier comprises a moiety that targets the myocardium.

DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once the DNA is isolated, it can be placed into an expression vector, such as that disclosed in PCT publication No. WO87/04462, and the expression vector can then be transfected into host cells that do not otherwise produce immunoglobulin, such as e.coli (e.coli) cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells, for synthesis of monoclonal antibodies in the recombinant host cells. See, for example, PCT publication No. WO 87/04462. The DNA may also be modified, for example, by replacing the homologous murine sequences with the coding sequences for human heavy and light chain constant domains, Morrison et al, proc.nat.acad.sci.81:6851,1984, or by covalently linking all or a portion of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, "chimeric" or "hybrid" antibodies having the binding specificity of the monoclonal antibodies herein are prepared.

Methods known in the art can be used to identify or characterize CD70 antibodies as described herein, whereby a decrease in the expression level of CD70 is detected and/or measured. In some embodiments, the CD70 antibody is identified by incubating the candidate agent with CD70 and monitoring the concomitant decrease in binding and/or expression levels of CD 70. Binding assays may be performed with one or more purified CD70 polypeptides or with cells naturally expressed or transfected to express one or more CD70 polypeptides. In one embodiment, the binding assay is a competitive binding assay in which the ability of a candidate antibody to compete for CD70 binding with a known CD70 antibody is assessed. The assay can be performed in various formats, including ELISA formats.

After preliminary identification, the activity of the candidate CD70 antibody may be further confirmed and refined by known bioassays for testing biological activity of interest. Alternatively, the bioassay may be used to directly screen candidates. Some methods of identifying and characterizing antibodies are described in detail in the examples.

The CD70 antibody can be characterized using methods well known in the art. For example, one approach is to identify the epitope or "epitope mapping" to which it binds. Many methods for mapping and characterizing the location of epitopes on proteins are known in the art, including resolution of the crystal structure of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,1999, Chapter 11. In another example, epitope mapping can be used to determine the sequence to which an antibody binds. Epitope mapping is commercially available from a variety of sources, such as Pepscan Systems (Edelhertweg 15,8219 PH Lelystad, The Netherlands). The epitope may be a linear epitope (i.e., contained within a single stretch of amino acids), or may be a conformational epitope formed by the three-dimensional interaction of amino acids that are not necessarily contained within a single stretch. Peptides of different lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinant) and used in binding assays with CD70 or other tumor antigen antibodies. In another example, the epitope to which the CD70 antibody binds can be determined in a systematic screen by using overlapping peptides derived from the CD70 sequence and determining the binding of the CD70 antibody. According to the gene fragment expression assay, the open reading frame encoding CD70 is fragmented randomly or by specific genetic construction, and the reactivity of the expressed fragment of CD70 with the antibody to be tested is determined. Gene fragments can be generated, for example, by PCR and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. Binding of the antibody to radiolabeled CD70 was then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In another example, mutagenesis of the antigen binding domain, domain exchange experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, a domain swapping experiment can be performed using mutant CD70, in which various fragments of the CD70 protein have been replaced (swapped) with CD70 from another species (e.g., mouse) or sequences of closely related but antigenically distinct proteins. The importance of a particular CD70 fragment for antibody binding can be assessed by assessing antibody binding to mutant CD 70. For antibodies specific for CD70 (i.e., antibodies that do not bind CD70wt (wild-type) or any other protein), an epitope can be inferred from the sequence alignment of CD70 to CD70 wt.

Yet another method that can be used to characterize the CD70 antibody is to determine whether the CD70 antibody binds to the same epitope as other antibodies using a competition assay for other antibodies (i.e., various fragments on CD70) that are known to bind to the same antigen. Competitive assays are well known to those skilled in the art.

The expression vector may be used to direct the expression of the CD70 antibody. One skilled in the art is familiar with the administration of expression vectors to obtain in vivo expression of exogenous proteins. See, for example, U.S. patent nos. 6,436,908, 6,413,942, and 6,376,471. Administration of the expression vector includes local or systemic administration, including injection, oral administration, particle gun or catheterization administration, and local administration. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglia, or to the coronary arteries, atria, ventricles or pericardium.

Targeted delivery of therapeutic compositions containing expression vectors or subgenomic polynucleotides may also be used. Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al, Trends biotechnol, 1993,11: 202; chiou et al, Gene Therapeutics, Methods And Applications Of Direct Gene Transfer, eds. J.A.Wolff, 1994; wu et al, J.biol.chem.,263:621,1988; wu et al, J.biol.chem.,269:542,1994; zenke et al, proc.natl.acad.sci.usa,87:3655,1990; and Wu et al, J.biol.chem.,266:338,1991. In gene therapy protocols, therapeutic compositions containing polynucleotides are administered topically in the range of about 100ng to about 200mg of DNA. Concentration ranges of about 500ng to about 50mg, about 1g to about 2mg, about 5g to about 500g, and about 20g to about 100g of DNA may also be used in gene therapy regimens. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy,1:51,1994; Kimura, Human Gene Therapy,5:845,1994; Connelly, Human Gene Therapy,1995,1: 185; and Kaplitt, Nature Genetics,6:148,1994). Endogenous mammalian or heterologous promoters can be used to induce expression of such coding sequences. Expression of the coding sequence may be constitutive or regulated.

For delivery of desired polynucleotides and expression of virus-based vectors in desired cells are well known in the art. Exemplary virus-based vectors include, but are not limited to, recombinant retroviruses (see, e.g., PCT publication Nos. WO 90/07936, WO 94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, WO 91/02805, U.S. Pat. Nos. 5,219,740 and 4,777,127, UK patent No. 2,200,651, and European patent No. 0345242), alphavirus-based vectors (e.g., Sindbis (Sindbis) viral vectors, Semliki (Semliki) forest viruses (ATCC VR-67; ATCC VR-1247), Ross river viruses (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis viruses (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT publication No. WO 94/12649, WO 94/03622; WO 93/25698; WO 94/03622; and European patent No. 0345242), WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus can also be used, as described by Curiel, hum.

Non-viral delivery vehicles and methods may also be employed, including but not limited to polycationic condensed DNA linked or not to a killed adenovirus alone (see, e.g., Curiel, hum. gene ther.,3:147,1992); ligand-linked DNA (see, e.g., Wu, j.biol.chem.,264:16985,1989); eukaryotic cells deliver vehicle cells (see, e.g., U.S. patent No. 5,814,482, PCT publication nos. WO 95/07994, WO 96/17072, WO 95/30763, and WO 97/42338) and the electrical neutralization or fusion of nucleic acids with the cell membrane. Naked DNA may also be used. Exemplary naked DNA introduction methods are described in PCT publication No. WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can be used as gene delivery vehicles are described in U.S. Pat. nos. 5,422,120; PCT publications WO 95/13796, WO 94/23697, WO 91/14445 and EP 0524968 are described. Additional methods are described in Philip, mol.cell biol.,14:2411,1994 and Wuffendin, Proc.Natl.Acad.Sci.,91:1581,1994.

In some embodiments, the invention encompasses compositions, including pharmaceutical compositions, comprising an antibody described herein or prepared by a method and having the features described herein. As used herein, a composition comprises one or more antibodies that bind to CD70, and/or one or more polynucleotides comprising sequences encoding one or more of these antibodies. These compositions may additionally comprise suitable excipients, such as pharmaceutically acceptable excipients, including buffers, which are well known in the art.

The invention also provides methods of making any of these antibodies. The antibodies of the invention can be prepared by procedures known in the art. The polypeptide may be produced by proteolytic or other degradation of the antibody, by recombinant methods as described above (i.e., single or fusion polypeptides), or by chemical synthesis. Polypeptides of antibodies, particularly shorter polypeptides of up to about 50 amino acids, can be conveniently prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, antibodies can be produced by an automated polypeptide synthesizer using solid phase methods. See also, U.S. Pat. nos. 5,807,715, 4,816,567, and 6,331,415.

In another alternative, the antibody may be recombinantly produced using procedures well known in the art. In one embodiment, the polynucleotide comprises sequence 31H1, 63B2, 40E3, 42C3, 45F11, 64F9, 72C2, 2F10, 4F11, 10H10, 17G6, 65E11, P02B10, P07D03, P08a02, P08E02, P08F08, P08G02, P12B09, P12F02, P12G07, P13F04, P15D02, or P16C05 encoding the heavy and/or light chain variable region of the antibody. The sequences encoding the antibody of interest may be maintained in a vector in a host cell, which may then be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

Heterologous conjugate antibodies comprising two covalently linked antibodies are also within the scope of the invention. Such antibodies have been used to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), as well as in the treatment of HIV infection (PCT publications WO 91/00360 and WO 92/200373, EP 03089). The heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable crosslinking agents and techniques are well known in the art and are described in U.S. Pat. No. 4,676,980.

Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate.

In recombinant humanized antibodies, the Fc portion may be modified to avoid interaction with Fc γ receptors as well as complement and immune systems. Techniques for making such antibodies are described in WO 99/58572. For example, if the antibody is used in clinical trials and treatments for humans, the constant region can be engineered to more closely resemble a human constant region to avoid an immune response. See, for example, U.S. patent nos. 5,997,867 and 5,866,692.

The invention encompasses modifications to the antibodies and polypeptides of the invention, including variants as shown in table 5, including functionally equivalent antibodies that do not significantly affect properties, as well as variants with increased or decreased activity and/or affinity. For example, the amino acid sequence may be mutated to obtain an antibody having a desired binding affinity for CD 70. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides having conservative substitutions of amino acid residues, one or more amino acid deletions or additions that do not significantly detrimentally alter functional activity, or mature (enhance) the affinity of the polypeptide for its ligand, or the use of chemical analogs.

Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues or other insertional variants of antibody molecules fused to epitope tags include the fusion of the N-terminus or C-terminus of the antibody with an enzyme or polypeptide, which increases the half-life of the antibody in blood circulation.

Substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted at that position. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in table 5 under the heading "conservative substitutions". More substantial changes, designated "exemplary substitutions" in table 5, or as described further below with reference to amino acid classes, can be introduced if such substitutions result in a change in biological activity, and the products screened. In some embodiments, a substitution variant of an antibody provided herein has no more than 15, 14, 13, 12, 11, 10, 9,8, 7, 6,5, 4, 3,2, or 1 conservative substitution in the VH or VL region as compared to a reference parent antibody. In some embodiments, the substitutions are not within a CDR of a VH or VL region.

Table 5: amino acid substitution

Substantial modification of antibody biological properties is achieved by selecting substitutions that differ significantly in their effect in maintaining the following properties: (a) the structure of the polypeptide backbone in the replacement region (e.g., a folded or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the side chain volume. Naturally occurring amino acid residues are divided into groups according to common side chain properties:

(1) non-polar: norleucine, Met, Ala, Val, Leu, Ile;

(2) the polarity is uncharged: cys, Ser, Thr, Asn, Gln;

(3) acidic (negatively charged): asp and Glu;

(4) basic (positively charged): lys, Arg;

(5) residues that influence chain orientation: gly, Pro; and

(6) aromatic: trp, Tyr, Phe, His.

Non-conservative substitutions are made by replacing a member of one of these classes with another class.

Any cysteine residues not involved in maintaining the proper conformation of the antibody may also be generally replaced with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Instead, one or more cysteine bonds may be added to the antibody to improve the stability of the antibody (particularly where the antibody is an antibody fragment, such as an Fv fragment).

Amino acid modifications can range from changing or modifying one or more amino acids to a redesign of the finished region (such as the variable region). Changes in the variable region may alter binding affinity and/or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are made within a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are made within a CDR domain. In still other embodiments, the CDR domain is CDR H3 and/or CDR L3.

Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, e.g., glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, chem. Immunol.65:111-128, 1997; Wright and Morrison, TibTECH 15:26-32,1997). The oligosaccharide side chains of immunoglobulins can influence the function of the protein (Boyd et al, mol. Immunol.32:1311-1318, 1996; Wittwe and Howard, biochem.29:4175-4180,1990) and the intramolecular interactions between parts of the glycoprotein, which can influence the conformation and provide a three-dimensional surface of the glycoprotein (Jefferis and Lund, supra; Wys and Wagner, Current Opin. Biotech.7:409-416, 1996). Oligosaccharides can also be used to target a given glycoprotein to certain molecules based on a particular recognition structure. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). Specifically, CHO cells with tetracycline-regulated expression of β (1,4) -N-acetylglucosaminyltransferase III (GnTIII) (a glycosyltransferase that catalyzes the formation of bisecting GlcNAc) have been reported to have improved ADCC activity (Umana et al, Mature Biotech.17:176-180, 1999).

Glycosylation of antibodies is usually N-linked or O-linked. N-linkage refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain, where X is any amino acid except proline. Thus, the presence of any of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of a sugar (i.e., one of N-acetylgalactosamine, galactose, or xylose) to a hydroxyamino acid (most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used).

The addition of glycosylation sites to the antibody is conveniently achieved by altering the amino acid sequence such that the antibody contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Alterations (for O-linked glycosylation sites) may also be made by the addition of one or more serine or threonine residues to the sequence of the original antibody or by substitution with one or more serine or threonine residues.

The glycosylation pattern of an antibody can also be altered without altering the base nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins (e.g., antibodies) as potential therapeutic agents is rarely a native cell, variations in the glycosylation pattern of antibodies can be expected (see, e.g., Hse et al, J.biol.chem.272: 9062-.

Factors that influence glycosylation during recombinant production of antibodies include, in addition to host cell selection, growth pattern, medium formulation, culture density, oxygenation, pH, purification protocols, and the like. Various approaches have been proposed to alter the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (U.S. Pat. nos. 5,047,335, 5,510,261 and 5,278,299). Glycosylation or certain types of glycosylation can be enzymatically removed from glycoproteins, for example, using endoglycosidase h (endo h), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. In addition, recombinant host cells can be genetically engineered to be deficient in processing certain types of polysaccharides. These and similar techniques are well known in the art.

Other methods of modification include the use of coupling techniques known in the art, including but not limited to enzymatic means, oxidative displacement, and chelation. Modifications can be used, for example, for attachment of labels to perform immunoassays. Modified polypeptides are prepared using procedures established in the art, and the polypeptides can be screened using standard assays known in the art, some of which are described below and in the examples.

Other antibody modifications include antibodies modified as described in PCT publication No. WO 99/58572. In addition to the binding domain for the target molecule, these antibodies comprise an effector domain having an amino acid sequence that is substantially homologous to all or a portion of the constant region of a human immunoglobulin heavy chain. These antibodies are capable of binding to a target molecule without triggering significant complement-dependent lysis or cell-mediated destruction of the target. In some embodiments, the effector domain is capable of specifically binding FcRn and/or fcyriib. These are generally based on heavy chains C derived from two or more human immunoglobulin heavy chains_H2 domain. Antibodies modified in this manner are particularly useful in chronic antibody therapy to avoid inflammatory and other adverse effects on conventional antibody therapy.

The present invention includes affinity maturation embodiments. For example, affinity matured antibodies can be generated by procedures known in the art (Marks et al, Bio/Technology,10:779- & 783, 1992; Barbas et al, Proc Nat. Acad. Sci, USA 91:3809- & 3813, 1994; Schier et al, Gene,169:147- & 155, 1995; Yelton et al, J.Immunol.,155:1994- & 2004, 1995; Jackson et al, J.Immunol.,154(7): 0- & 9,1995, Hawkins et al, J.mol.biol.,226:889- & 896, 1992; and PCT publication No. WO 2004/058184).

The following methods can be used to adjust the affinity and characterize the CDRs of the antibody. One method of characterizing CDRs of an antibody and/or altering (such as improving) the binding affinity of a polypeptide (such as an antibody) is referred to as "library scanning mutagenesis". In general, the working principle of library scanning mutagenesis is as follows. One or more amino acid positions in the CDRs are replaced with two or more (such as 3,4, 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20) amino acids using art-recognized methods. This results in small clone libraries (in some embodiments, one clone per amino acid position analyzed), each library having a complexity of two or more members (if two or more amino acids are substituted at each position). Typically, libraries also include clones containing the natural (unsubstituted) amino acids. From a small number of grams from each libraryClones (e.g., about 20-80 clones (depending on the complexity of the library)) are screened for binding affinity to the target polypeptide (or other binding target) and candidates with increased, identical, reduced, or no binding are identified. Methods for determining binding affinity are well known in the art. Biacore may be used^TMSurface plasmon resonance analysis to determine binding affinity, the analysis can detect about 2 times or more binding affinity difference. When the starting antibody has been raised to a relatively high affinity (e.g., a K of about 10nM or less)_D) Upon binding, Biacore^TMIt is particularly useful. Embodiments describe herein the use of Biacore^TMScreening by surface plasmon resonance.

Binding affinity can be determined using Kinexa biosensors, scintillation proximity assays, ELISA, ORIGEN Immunoassays (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display. Binding affinities may also be screened using a suitable bioassay.

In some embodiments, each amino acid position in the CDRs is replaced (in some embodiments, one at a time) with all 20 natural amino acids using art-recognized mutagenesis methods (some of which are described herein). This results in small clone libraries (in some embodiments, one clone per amino acid position analyzed), each library having a complexity of 20 members (if all 20 amino acids are substituted at each position).

In some embodiments, the library to be screened comprises substitutions in two or more positions, which substitutions may be in the same CDR or in two or more CDRs. Thus, a library may comprise substitutions in two or more positions in one CDR. The library may comprise substitutions in two or more positions in two or more CDRs. The library may comprise substitutions in 3,4, 5 or more positions that are present in two, three, four, five or six CDRs. Substitutions can be made using low redundancy codons. See, e.g., Table 2 of Balint et al, Gene 137(1): 109-.

The CDRs may be CDRH3 and/or CDRL 3. The CDRs may be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH 3. The CDRs may be Kabat CDRs, Chothia CDRs, or extended CDRs.

Candidates with improved binding can be sequenced to identify CDR substitution mutants that produce improved affinity (also referred to as "improved" substitutions). The bound candidates can also be sequenced to identify CDR substitutions that retain binding.

Multiple rounds of screening can be performed. For example, candidates with improved binding (each candidate comprising an amino acid substitution at one or more positions of one or more CDRs) can also be used in the design of a second library containing at least the original and substituted amino acids at each improved CDR position (i.e., substitution mutants that exhibit improved binding at amino acid positions in CDRs). The preparation and screening or selection of the library is discussed further below.

Library scanning mutagenesis also provides a means to characterize the CDRs so that cloning frequency with improved binding, identical binding, reduced binding or no binding also provides information concerning the importance of each amino acid position for the stability of the antibody-antigen complex. For example, if a position of a CDR retains binding when changed to all 20 amino acids, that position is identified as unlikely to be a position required for antigen binding. Conversely, if a position of a CDR remains bound for only a small fraction of substitutions, that position is identified as a position that is important to the function of the CDR. Thus, library scanning mutagenesis methods yield information about positions in the CDRs that can be changed to many different amino acids (including all 20 amino acids), as well as positions in the CDRs that cannot be changed or can only be changed to a few amino acids.

Candidates with improved affinity may be combined in a second library comprising the improved amino acid, the original amino acid at that position, and, depending on the complexity of the library desired or allowed, may additionally comprise additional substitutions at that position using the desired screening or selection method. Furthermore, adjacent amino acid positions can be randomly grouped into at least two or more amino acids, if desired. Random grouping of adjacent amino acids may allow additional conformational flexibility to be created in the mutant CDRs, which in turn may allow or facilitate the introduction of a number of improved mutations. The library may also comprise substitutions at positions that do not show improved affinity in the first round of screening.

Screening or selecting library members having improved and/or altered binding affinities from the second library using any method known in the art, including using Biacore^TMScreening by surface plasmon resonance analysis, and selection using any selection method known in the art, including phage display, yeast display, and ribosome display.

The invention also encompasses fusion proteins comprising one or more fragments or regions from an antibody of the invention. In one embodiment, a fusion polypeptide is provided comprising at least 10 contiguous amino acids of the variable light chain region set forth in SEQ ID NO 1,3, 5,7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47, and/or at least 10 amino acids of the variable heavy chain region set forth in SEQ ID NO 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48. In other embodiments, a fusion polypeptide is provided comprising at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 consecutive amino acids of the variable light chain region and/or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 consecutive amino acids of the variable heavy chain region. In another embodiment, the fusion polypeptide comprises one or more CDRs. In still other embodiments, the fusion polypeptide comprises the CDR H3(VH CDR3) and/or CDR L3(VL CDR 3). For the purposes of the present invention, a fusion protein contains one or more antibodies and another amino acid sequence to which it is not attached in the native molecule (e.g., a heterologous sequence or a homologous sequence from another region). Exemplary heterologous sequences include, but are not limited to, a "tag," such as a FLAG tag or a 6His tag (SEQ ID NO: 531). Labels are well known in the art.

Fusion polypeptides may be produced by methods known in the art, such as synthetic or recombinant production. Typically, the fusion proteins of the invention are prepared by expressing polynucleotides encoding them using recombinant methods described herein, but they can also be prepared by other means known in the art, including, for example, chemical synthesis.

The invention also provides compositions comprising an antibody conjugated (e.g., linked) to an agent that facilitates coupling to a solid support, such as biotin or avidin. For the sake of brevity, reference will be made to antibodies in their entirety, with the understanding that these methods are applicable to any of the CD70 antibody embodiments described herein. Conjugation generally refers to linking these components as described herein. The attachment can be achieved in a variety of ways (typically by holding the components together tightly at least in terms of administration). For example, when the reagent and the antibody each have a substituent capable of reacting with each other, a direct reaction between the reagent and the antibody may be performed. For example, a nucleophilic group on one of them (such as an amino or mercapto group) can react with a carbonyl-containing group on the other of them (such as an anhydride or acid halide), or with an alkyl group containing a good leaving group (e.g., halide).

The invention also provides isolated polynucleotides encoding the antibodies of the invention, as well as vectors and host cells comprising the polynucleotides.

Accordingly, the present invention provides a polynucleotide (or composition, including a pharmaceutical composition) comprising a polynucleotide encoding any one of: 31H1, 63B2, 40E3, 42C3, 45F11, 64F9, 72C2, 2F10, 4F11, 10H10, 17G6, 65E11, P02B10, P07D03, P08a02, P08E02, P08F08, P08G02, P12B09, P12F02, P12G07, P13F04, P15D02, or P16C05, or any fragment or portion thereof having the ability to bind CD 70.

In another aspect, the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) and polypeptides described herein, such as antibodies and polypeptides having impaired effector function. Polynucleotides can be prepared and expressed by procedures known in the art.

In another aspect, the invention provides a composition (such as a pharmaceutical composition) comprising any one of the polynucleotides of the invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any one of the antibodies described herein.

The administration of the expression vector and polynucleotide composition is further described herein.

In another aspect, the invention provides a method of making any of the polynucleotides described herein.

Polynucleotides complementary to any such sequence are also encompassed by the invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one manner, as well as mRNA molecules that do not contain introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the invention, and the polynucleotides may, but need not, be linked to other molecules and/or support materials.

The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions, and/or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the naturally-occurring immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, still more preferably at least about 90% identity, and most preferably at least about 95% identity to the polynucleotide sequence encoding the native antibody or a portion thereof.

Two nucleotide or amino acid sequences are considered "identical" if the sequences of nucleotides or amino acids in the two sequences are identical when aligned for maximum correspondence as described below. Comparisons between two sequences are typically made by comparing the sequences over a comparison window, thereby identifying and comparing local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20, typically 30 to about 75, or 40 to about 50 consecutive positions in which a sequence can be compared to a reference sequence having the same number of consecutive positions after optimal alignment of the two sequences.

Sequences can be optimally aligned for comparison using default parameters using the Megalign program in the Lasergene bioinformatics software package (DNASTAR, inc., Madison, WI). This program is embodied by several alignment schemes described in the following references: dayhoff, M.O.,1978, A model of evolution change in proteins-substrates for detecting displacement relationships, described in Dayhoff, M.O, (ed.) Atlas of Protein sequences and structures, National biological Research Foundation, Washington DC, Vol.5, supplement 3, p.345 and 358; hein J.,1990, Unified Approach to Alignment and olefins, Methods in Enzymology, pp.626-; higgins, D.G. and Sharp, P.M.,1989, CABIOS 5: 151-; myers, E.W. and Muller W.,1988, CABIOS 4: 11-17; robinson, E.D.,1971, comb. Theor.11: 105; santou, N., Nes, M.,1987, mol.biol.Evol.4: 406-425; sneath, p.h.a. and Sokal, r.r.,1973, Numerical taxomones and Practice of Numerical taxomones, Freeman Press, San Francisco, CA; wilbur, W.J., and Lipman, D.J.,1983, Proc. Natl. Acad. Sci. USA 80: 726-.

Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein for optimal alignment of the two sequences, a portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20% or less, typically 5% to 15%, or 10% to 12%, as compared to the reference sequence (which does not comprise additions or deletions). The percentages are calculated by the following method: the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences is determined to give the number of matched positions, the number of matched positions is divided by the total number of positions in the reference sequence (i.e., the window size), and the result is multiplied by 100 to give the percentage of sequence identity.

The variant may additionally or alternatively be substantially homologous to the native gene or a portion thereof or to the complement. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence (or complementary sequence) encoding a natural antibody.

Suitable "moderately stringent conditions" include a pre-wash in a solution of 5 XSSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridization at 50-65 ℃ under 5 XSSC overnight; then washed twice with 2X, 0.5X and 0.2 XSSC containing 0.1% SDS, respectively, at 65 ℃ for 20 minutes.

As used herein, "high stringency conditions" or "high stringency conditions" refer to: (1) washing with low ionic strength and high temperature, e.g. 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate, at 50 ℃; (2) denaturing reagents such as formamide, e.g., 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer, pH 6.5 and 750mM sodium chloride, 75mM sodium citrate, at 42 ℃ are used during hybridization; or (3) washing with 50% formamide, 5 XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 XDenhart (Denhardt) solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS and 10% dextran sulfate in 0.2 XSSC (sodium chloride/sodium citrate) at 42 ℃ and in 50% formamide at 55 ℃ followed by a high stringency wash at 55 ℃ comprising 0.1 XSSC with EDTA. The skilled person will recognize how to adjust the temperature, ionic strength, etc. as required to accommodate factors such as probe length, etc.

One of ordinary skill in the art will appreciate that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the invention. An allele is an endogenous gene that is altered by one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques, such as hybridization, amplification, and/or database sequence comparison.

The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One skilled in the art can use the sequences provided herein and a commercial DNA synthesizer to generate the desired DNA sequence.

For preparation of polynucleotides using recombinant methods, polynucleotides comprising the desired sequences can be inserted into a suitable vector, which in turn can be introduced into a suitable host cell for replication and amplification, as discussed further herein. The polynucleotide may be inserted into the host cell by any means known in the art. Cells are transformed by direct uptake, endocytosis, transfection, F-mating, or electroporation by introduction of exogenous polynucleotides. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrating vector (such as a plasmid), or integrated into the host cell genome. The polynucleotides so amplified can be isolated from the host cell by methods well known in the art. See, e.g., Sambrook et al, 1989.

Alternatively, PCR allows for the replication of DNA sequences. PCR techniques are well known in The art and are described in U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, and PCR, The Polymerase Chain Reaction, eds by Mullis et al, Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vector and inserting the vector into an appropriate host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, as described, for example, in Sambrook et al, 1989, supra.

Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. Although the cloning vector selected may vary depending on the host cell desired for use, useful cloning vectors are generally self-replicating, may have a single target of a particular restriction endonuclease, and/or may carry a gene for a marker that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK +) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors (such as pSA3 and pAT 28). These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene and Invitrogen.

Expression vectors are generally replicable polynucleotide constructs comprising a polynucleotide according to the invention. This means that the expression vector must be replicable in the host cell either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenovirus, adeno-associated virus, retrovirus), cosmids, and one or more of the expression vectors disclosed in PCT publication No. WO 87/04462. The carrier components may generally include, but are not limited to, one or more of the following components: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational control elements, such as a ribosome binding site, a translation initiation site, and a stop codon, are also typically required.

The vector containing the polynucleotide of interest may be introduced into the host cell by any of a number of suitable means, including electroporation, transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; bombardment of particles; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of vector or polynucleotide to introduce will generally depend on the characteristics of the host cell.

The invention also provides a host cell comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating a gene encoding an antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotes such as e.coli or bacillus subtilis and yeasts such as saccharomyces cerevisiae (s.cerevisae), schizosaccharomyces pombe (s.pombe) or kluyveromyces lactis (k.lactis). Preferably, the host cell expresses the cDNA at a level that is about 5-fold higher, more preferably 10-fold higher, even more preferably 20-fold higher than the level of the corresponding endogenous antibody or protein of interest (if present) in the host cell. Host cells are screened for specific binding to CD70 by immunoassay or FACS. Cells that overexpress the antibody or protein of interest can be identified.

Methods of use of CD70 antibodies

The antibodies of the invention can be used in a variety of applications, including but not limited to therapeutic and diagnostic treatment methods.

The antibodies (e.g., monospecific and bispecific) obtained by the methods described above can be used as medicaments. In some embodiments, such a medicament may be used to treat cancer. In some embodiments, the cancer is a cancer of hematopoietic origin, such as lymphoma or leukemia. In some embodiments, the cancer is renal cell carcinoma, glioblastoma, glioma such as low grade glioma, non-hodgkin's lymphoma (NHL), Hodgkin's Disease (HD), waldenstrom's macroglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

In some embodiments, a method of inhibiting tumor growth or progression in a subject having a malignant cell that expresses CD70 is provided, comprising administering to a subject in need thereof an effective amount of a composition comprising a CD70 antibody (e.g., a CD70-CD3 bispecific antibody) as described herein. In other embodiments, a method of inhibiting metastasis of a cell expressing CD70 in a subject is provided, comprising administering to a subject in need thereof an effective amount of a composition comprising a CD70 antibody (e.g., a CD70-CD3 bispecific antibody) as described herein. In other embodiments, a method of inducing tumor regression in malignant cells in a subject is provided, comprising administering to a subject in need thereof an effective amount of a composition comprising a CD70 antibody (e.g., a CD70-CD3 bispecific antibody) as described herein.

In some embodiments, an antibody according to the invention (e.g., a CD70-CD3 bispecific antibody) can be used in the manufacture of a medicament for treating cancer in a patient in need thereof.

In some embodiments, the treatment may be combined with one or more therapies for cancer selected from the group consisting of: antibody therapy, chemotherapy, cytokine therapy, targeted therapy, vaccine therapy, dendritic cell therapy, gene therapy, hormone therapy, surgical resection, laser therapy, and radiation therapy.

For example, in some embodiments, the CD70 antibodies of the invention (e.g., the CD70-CD3 bispecific antibody) are administered to a patient in combination with (e.g., prior to, concurrently with, or subsequent to) treatment with a small molecule Tyrosine Kinase Inhibitor (TKI), such as sunitinib and pazopanib, which target Vascular Endothelial Growth Factor (VEGF) receptors, a VEGF-targeting monoclonal antibody, such as bevacizumab, a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, and a high dose of IL-2. In some embodiments, the CD70 antibodies of the invention (e.g., CD70-CD3 bispecific antibodies) are administered to a patient in combination with one or more of the following antibodies: an anti-PD-1 antibody (e.g., nivolumab, palivizumab, or PF-06801591), an anti-PD-L1 antibody (e.g., avizumab, atuzumab, or dervacizumab), an anti-OX 40 antibody (e.g., PF-04518600), an anti-4-1 BB antibody (e.g., PF-05082566), an anti-MCSF antibody (e.g., PD-0360324), an anti-GITR antibody, and/or an anti-TIGIT antibody.

Administration of an antibody (e.g., monospecific or bispecific) according to the invention may be carried out in any convenient manner, including by nebulization inhalation, injection, ingestion, blood transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intracranially, intranodal, intramedullary, intramuscular, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the antibody composition of the invention is preferably administered by intravenous injection.

In some embodiments, administration of the antibody (e.g., monospecific or bispecific) may include administration of, for example, about 0.01 to about 20mg/kg body weight, including all integer values of mg/kg within these ranges. In some embodiments, administration of the antibody may comprise administration of about 0.1 to 10mg/kg body weight, including all integer values of mg/kg within these ranges. The antibody may be administered in one or more doses. In some embodiments, the effective amount of the antibody can be administered in a single dose. In some embodiments, the effective amount of the antibody may be administered in more than one dose over a period of time. The timing of administration is determined by the attending physician and depends on the clinical condition of the patient. Although individual requirements vary, determination of an optimal range for an effective amount of a given antibody (e.g., monospecific or bispecific) for a particular disease or disorder is within the skill of the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dose administered will depend on the age, health and weight of the recipient, the type of concurrent treatment (if any), the frequency of treatment and the nature of the effect desired. In some embodiments, an effective amount of heteromultimeric antibodies or compositions comprising those antibodies are administered parenterally. In some embodiments, the administration may be intravenous administration. In some embodiments, administration can be performed directly by injection within the tumor.

In some embodiments, the anti-CD 70 antibodies provided herein can be used for diagnostic purposes, such as assays for identifying CD70 protein in a sample (e.g., an immunohistochemical assay) or patient.

Composition comprising a metal oxide and a metal oxide

In one aspect, the invention provides a pharmaceutical composition comprising an antibody (e.g., monospecific or bispecific) of the invention, or a portion thereof, as described above, dissolved in a pharmaceutically acceptable carrier. In certain embodiments, the polypeptides of the invention may exist in a neutral form (including zwitterionic forms) or as positively or negatively charged species. In some embodiments, the polypeptides may be complexed with counterions to form "pharmaceutically acceptable salts," which refers to complexes comprising one or more polypeptides and one or more counterions derived from pharmaceutically acceptable inorganic and organic acids and bases.

The antibody (e.g., monospecific or bispecific) or portion thereof may be administered alone or in combination with one or more other polypeptides of the invention or in combination with one or more other drugs (or any combination thereof). Accordingly, the pharmaceutical compositions, methods and uses of the invention also encompass embodiments in combination (co-administration) with other active agents, as detailed below.

As used herein, the terms "co-administration," "co-administration," and "in conjunction with … …" refer to the antibody of the invention and one or more other therapeutic agents, and are intended to mean and refer to and include the following: (i) when these components are formulated together as a single dosage form, such combination of an antibody disclosed herein and one or more therapeutic agents is administered to a patient in need of treatment, said single dosage form releasing the components to the patient substantially simultaneously; (ii) when the components are formulated separately from each other as separate dosage forms, such combination of an antibody disclosed herein and one or more therapeutic agents is administered substantially simultaneously to a patient in need of treatment, the separate dosage forms being taken substantially simultaneously by the patient such that the components are released substantially simultaneously to the patient; (iii) when these components are formulated separately from each other as separate dosage forms, such combination of an antibody disclosed herein and one or more therapeutic agents is administered sequentially to a patient in need of treatment, the separate dosage forms being ingested by the patient at significant time intervals between each administration over a continuous period of time, whereby the components are released to the patient at substantially different times; and (iv) when these components are formulated together as a single dosage form, such combination of the antibody disclosed herein and one or more therapeutic agents is administered sequentially to a patient in need of treatment, said single dosage form releasing the components in a controlled manner such that they are released to the patient simultaneously, sequentially and/or overlapping at the same and/or different times, wherein each portion may be administered by the same or different routes.

In general, the antibodies (e.g., monospecific or bispecific) or portions thereof disclosed herein are suitable for administration as a formulation in combination with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than one or more compounds of the present invention. The choice of excipient or excipients will depend to a large extent on factors such as the particular mode of administration, the effect of the excipients on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Further examples of pharmaceutically acceptable substances are wetting or minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which may extend the shelf-life or effectiveness of the antibody.

The pharmaceutical composition of the present invention and the method for preparing the same will be apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19 th edition (Mack Publishing Company, 1995). The pharmaceutical composition is preferably manufactured under GMP conditions.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in bulk as a single unit dose or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of active ingredient. The amount of active ingredient is typically equal to the dose of active ingredient to be administered to the subject or a suitable fraction of such dose, e.g. half or one third of such dose. Any method of administering peptides, proteins or antibodies accepted in the art may be suitably used for the heterodimeric proteins and their portions disclosed herein.

The pharmaceutical compositions of the present invention are generally suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical disruption of the tissue of the subject as well as administration of the pharmaceutical composition by disruption of the tissue, thus typically resulting in direct administration to the bloodstream, muscle, or internal organs. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, administration of the composition through a surgical incision, administration of the composition through a non-surgical wound penetrating tissue, and the like. In particular, parenteral administration is contemplated including, but not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusion; and renal dialysis infusion techniques. Preferred embodiments include intravenous and subcutaneous routes.

Formulations of pharmaceutical compositions suitable for parenteral administration typically comprise the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged or sold in a form suitable for bolus administration or continuous administration. Formulations for injection may be prepared, packaged or sold in unit dosage form, such as in ampoules or in multi-dose containers with a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may additionally comprise one or more additional ingredients, including but not limited to suspending, stabilizing or dispersing agents. In one embodiment of the formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffers (preferably at a pH of 3 to 9), but for some applications they may be more suitably formulated as sterile nonaqueous solutions or dry forms which may be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions (e.g., aqueous propylene glycol or dextrose solutions). Such dosage forms may be suitably buffered if desired. Other useful parenterally administrable formulations include those comprising the active ingredient (in microcrystalline form or in a liposomal preparation). Formulations for parenteral administration may be formulated for immediate release and/or modified release. Modified release formulations include controlled release, delayed release, sustained release, pulsed release, targeted release and programmed release formulations. For example, in one aspect, a sterile injectable solution can be prepared by the following steps: the desired amount of heterodimeric protein (e.g., bispecific antibody) is incorporated with one or a combination of ingredients enumerated above in an appropriate solvent, as needed, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

The dosage regimen may be adjusted to provide the best desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be reduced or increased proportionally according to the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration of the dosage and homogeneity. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the patients/subjects to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention is generally determined by and directly dependent on the following factors: (a) the unique characteristics of chemotherapeutic agents and the particular therapeutic or prophylactic effect to be achieved, and (b) limitations inherent in the art of compounding such active compounds to treat the sensitivity of an individual.

Thus, in light of the disclosure provided herein, the skilled artisan will appreciate that the dosage and dosing regimen may be adjusted according to methods well known in the therapeutic arts. That is, the maximum tolerated dose can be readily established, and an effective amount to provide a detectable therapeutic benefit to the patient can also be determined, and the timing requirements for administering each agent to provide a detectable therapeutic benefit to the patient can be determined. Thus, while certain dosages and administration regimens are exemplified herein, these examples in no way limit the dosages and administration regimens that may be provided to a patient in practicing the invention.

Notably, dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is also to be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen of the compositions of the invention can be based on a variety of factors including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration and the particular antibody employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. For example, the dosage may be adjusted according to pharmacokinetic or pharmacodynamic parameters, which may include clinical effects, such as toxic effects and/or laboratory values. Thus, the present invention encompasses intra-patient dose escalation as determined by the skilled artisan. Determination of appropriate dosages and regimens is well known in the relevant art, and once provided with the teachings disclosed herein, the assays will be understood to be encompassed by the skilled artisan.

In general, for administration of the antibodies (monospecific or bispecific) described herein, candidate doses can be administered daily, weekly, every other week, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every ten weeks, every twelve weeks, or more than every twelve weeks. For repeated administrations over several days or longer, depending on the condition, the treatment is maintained until the desired suppression of symptoms occurs or until a sufficient level of treatment is achieved, e.g., to alleviate symptoms associated with cancer. The progress of the therapy can be readily monitored by conventional techniques and assays. The dosing regimen, including the anti-FLT monospecific or bispecific antibody used, may vary over time.

In some embodiments, the candidate dose is administered daily at a dose ranging from any of about 1 to 30 to 300 to 3mg/kg, to 30mg/kg, to 100mg/kg or more, depending on the factors mentioned above. For example, daily doses of about 0.01mg/kg, about 0.03mg/kg, about 0.1mg/kg, about 0.3mg/kg, about 1mg/kg, about 2.5mg/kg, about 3mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg and about 25mg/kg may be used.

In some embodiments, the candidate dose is administered weekly at a dose ranging from any of about 1 to 30 to 300 to 3mg/kg, to 30mg/kg, to 100mg/kg or more, depending on the factors mentioned above. For example, weekly dosages of about 0.01mg/kg, about 0.03mg/kg, about 0.1mg/kg, about 0.3mg/kg, about 0.5mg/kg, about 1mg/kg, about 2.5mg/kg, about 3mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 25mg/kg and about 30mg/kg may be used.

In some embodiments, the candidate dose is administered at a dose ranging from any of about 1 to 30 to 300 to 3mg/kg, to 30mg/kg, to 100mg/kg or more every two weeks, depending on the factors mentioned above. For example, a bi-weekly dose of about 0.1mg/kg, about 0.3mg/kg, about 1mg/kg, about 2.5mg/kg, about 3mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 25mg/kg and about 30mg/kg may be used.

In some embodiments, the candidate dose is administered at a dose ranging from any of about 1 to 30 to 300 to 3mg/kg, to 30mg/kg, to 100mg/kg or more every three weeks, depending on the factors mentioned above. For example, every three week doses of about 0.1mg/kg, about 0.3mg/kg, about 1mg/kg, about 2.5mg/kg, about 3mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg and about 50mg/kg may be used.

In some embodiments, the candidate dose is administered monthly or every four weeks at a dose ranging from any of about 1 to 30 to 300 to 3mg/kg, to 30 to 100mg/kg or more, depending on the factors mentioned above. For example, monthly doses of about 0.1mg/kg, about 0.3mg/kg, about 1mg/kg, about 2.5mg/kg, about 3mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, and about 50mg/kg may be used.

In other embodiments, the candidate dose is administered daily at a dose ranging from about 0.01mg to about 1200mg or more, depending on the factors mentioned above. For example, a daily dose of about 0.01mg, about 0.1mg, about 1mg, about 10mg, about 50mg, about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, or about 1200mg may be used.

In other embodiments, the candidate dose is administered weekly at a dose ranging from about 0.01mg to about 2000mg or more, depending on the factors mentioned above. For example, a weekly dose of about 0.01mg, about 0.1mg, about 1mg, about 10mg, about 50mg, about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, or about 2000mg may be used.

In other embodiments, the candidate dose is administered at a dose ranging from about 0.01mg to about 2000mg or more every two weeks, depending on the factors mentioned above. For example, a biweekly dose of about 0.01mg, about 0.1mg, about 1mg, about 10mg, about 50mg, about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, or about 2000mg may be used.

In other embodiments, the candidate dose is administered at a dose ranging from about 0.01mg to about 2500mg or more every three weeks, depending on the factors mentioned above. For example, every three weeks of a dose of about 0.01mg, about 0.1mg, about 1mg, about 10mg, about 50mg, about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, or about 2500mg may be used.

In other embodiments, the candidate dose is administered at a dose ranging from about 0.01mg to about 3000mg or more every four weeks or month, depending on the factors mentioned above. For example, a monthly dose of about 0.01mg, about 0.1mg, about 1mg, about 10mg, about 50mg, about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, about 2500, about 2600mg, about 2700mg, about 2800mg, about 2900mg, or about 3000mg may be used.

Reagent kit

The invention also provides kits for use in the method. Kits of the invention comprise one or more containers comprising an antibody (e.g., monospecific or bispecific) as described herein and instructions for use according to any of the methods of the invention described herein. In general, these instructions include descriptions of the administration of antibody proteins for the therapeutic treatments described above.

Instructions as described herein relating to the use of the antibody (e.g., monospecific or bispecific) generally include information about the dosage, schedule of administration, and route of administration of the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a sub-unit dose. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

The kit of the invention is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packages for use in combination with a particular device, such as an inhaler, nasal administration device (e.g., nebulizer) or infusion device (such as a micropump), are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a bispecific antibody. The container may additionally comprise a second pharmaceutically active agent.

The kit may optionally provide additional components such as buffers and explanatory information. Typically, a kit includes a container and a label or package insert on or associated with the container.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Examples

Example 1: determination of the kinetics and affinity of the human CD70/CD70 antibody interaction at 37 ℃

The kinetics and affinity of the anti-CD 70 antibodies disclosed herein can be measured on a Biacore T200 surface plasmon resonance biosensor (GE Lifesciences, Piscataway NJ).

Example 2: t-cell mediated killing of RCC cell lines in vitro using CD70-CD3 bispecific IgG

Human anti-CD 70 and human anti-CD 3(H2B4-VH-hnps VL-TK ("H2B 4")) antibodies are represented by human IgG2dA _ D265A, engineered with EEEE on one arm and RRRR on the other arm to perform a bispecific exchange at positions 223, 225 and 228 in the hinge region (e.g., (C223E or C223R), (E225E or E225R), and (P228E or P228R)) and at position 409 or 368 in the CH3 region of human IgG2(SEQ ID NO:279) (e.g., K409R or L368E (EU numbering scheme)). The CD70/CD3 bispecific antibody also has a mutation from D to A at position 265 (EU numbering scheme).

CD3+ T cells from human PBMCs were negatively selected using the human Pan T cell isolation kit (Miltenyi, San Diego CA). Targets expressing (786-O) cells and CD3+ T cells were plated at 20000 and 100000 cells/well, respectively, onto clear U-shaped bottom plates. Cells were treated with 8-fold serial dilutions of bispecific antibody. At 24 hours post-treatment, RCC cell depletion was determined by flow cytometry analysis. Cell depletion was measured by comparison with control-treated cells. EC50 was calculated by Prism software.

Example 3: CD70-CD3 bispecific IgG induce tumor ablation in RCC subcutaneous xenograft models

NOD Scid Gamma (NSG) mice were subcutaneously implanted with 786-O tumors once they reached 200mm³I.e., 2000 million expanded T cells per mouse were injected intraperitoneally. After two days, the anti-CD 70 bispecific antibody was injected intravenously at 300, 100, or 30ug/mL via tail vein injection to determine the optimal bispecific antibody dose.

Materials and methods:

NOD scid γ (NSG) mice were shaved and prepared for subcutaneous tumor implantation in the right flank. 786-O tumor cells known to express CD70 were expanded in RPMI supplemented with 10% FBS. On the 0 th day, the day,786-O cells were resuspended in serum-free RPMI at the desired concentration so that each animal was injected with 500 ten thousand cells. Each animal was injected subcutaneously with tumor cells dissolved in 100uL serum free RPMI combined with 100uL matrigel (Corning). Day 0 baseline body weights were recorded for all animals immediately after tumor implantation. Tumors were measured twice weekly using Digimatic calipers (Mitutoyo) starting on day 9 and body weights were recorded. On day 14, when the tumor reached 200mm³(standard error 8.39), 40 tumor-bearing mice were randomly divided into 4 groups of 10 mice each. T cells were thawed and expanded and then resuspended in serum-free RPMI at the desired concentration to inject 2000 million T cells per animal. Each animal was injected intraperitoneally with T cells dissolved in 200uL serum-free RPMI. After two days, bispecific antibody was injected intravenously via tail vein at doses of 300, 100 or 30ug/mL per animal. Tumors were measured weekly and body weights recorded twice until the untreated group reached the end of the study (1500 mm)³Tumor volume).

Tumor volumes (mean and error SEM) were plotted on GraphPad Prism and statistical data was calculated using one-way ANOVA and repeated measurements.

Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it should be understood that various changes and modifications may be made without departing from the teachings herein and the inventions claimed below. The foregoing embodiments are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings provided herein. While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily appreciate that many variations and modifications may be made to these exemplary embodiments without undue experimentation. All such variations and modifications are within the scope of the present teachings.

All references cited herein, including patents, patent applications, articles, texts, etc., and the references cited therein, are hereby incorporated by reference in their entirety to the extent they are not cited. If one or more of the incorporated documents and similar materials differ or contradict the present application, including but not limited to defined terms, usage of terms, described techniques, and the like, the present application controls.

The foregoing description and examples detail certain specific embodiments of the invention and describe the best mode contemplated by the inventors. It should be understood, however, that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.

Claims (23)

1. An isolated antibody that specifically binds to cluster of differentiation 70(CD70), wherein said antibody comprises

a) A heavy chain Variable (VH) region comprising: (i) VH complementarity determining region 1(CDR1) comprising SEQ ID NOs 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 406, 368, 369, 370, 368, 375, 392, 380, 376, 382, 398, 393, 410, 416, 220, 2, 382, 393, 2, 393, 410, 393, and 393, 410, 422. 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459, or 460; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 449, 450, 455, 456, 461, or 462; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457 or 463; and/or

b) A light chain Variable (VL) region comprising: (i) a VL CDR1 comprising the sequence set forth in SEQ ID NO 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, or 527; (ii) a VL CDR2 comprising a sequence set forth in SEQ ID NO 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, or 528; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526 or 529.

2. An isolated antibody that specifically binds to cluster of differentiation 70(CD70), wherein said antibody comprises:

a) a VH region comprising a VH CDR1, VH CDR2 and VH CDR3 of the VH sequence set forth in SEQ ID NOs 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329 or 331; and/or

b) A VL region comprising VL CDR1, VL 2, and VL CDR3 of the VL sequence set forth in SEQ ID NOs 1,3, 5,7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, or 330.

3. An isolated antibody that specifically binds to CD70 and competes with the antibody of claim 1.

4. A bispecific antibody, wherein the bispecific antibody is a full length antibody comprising a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen and a second antibody variable domain of the bispecific antibody that is capable of recruiting the activity of a human immune effector cell by specifically binding to an effector antigen localized on the human immune effector cell, wherein the first antibody variable domain comprises a heavy chain Variable (VH) region comprising SEQ ID NOs 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 16, 303, 305, 307, 309, 311, 313, 315, a, 317. 319, 321, 323, 325, 327, 329 or 331 of VH CDR1, VH CDR2 and VH CDR 3; and/or a light chain Variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NOs 1,3, 5,7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, or 330.

5. A bispecific antibody, wherein the bispecific antibody is a full length antibody comprising a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen, and comprising a second antibody variable domain of the bispecific antibody capable of recruiting the activity of a human immune effector cell by specifically binding to an effector antigen localized on the human immune effector cell, wherein the first antibody variable domain comprises

a) A heavy chain Variable (VH) region comprising: (i) VH complementarity determining region 1(CDR1) comprising SEQ ID NOs 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 406, 368, 369, 370, 368, 375, 392, 380, 376, 382, 398, 393, 410, 416, 220, 2, 382, 393, 2, 393, 410, 393, and 393, 410, 422. 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459, or 460; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 449, 450, 455, 456, 461, or 462; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457 or 463; and/or

b) A light chain Variable (VL) region comprising: (i) 93, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, or 527; (ii) a VL CDR2 comprising a sequence set forth in SEQ ID NO 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, or 528; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526 or 529.

6. The bispecific antibody of claim 5, wherein the second antibody variable domain specifically binds to the effector antigen CD 3.

7. The bispecific antibody of claim 6, wherein the second antibody variable domain comprises

a) A heavy chain Variable (VH) region comprising: (i) VH complementarity determining region 1(CDR1) comprising the sequence shown in SEQ ID NOs: 267, 268, or 269; (ii) VH CDR2 comprising the sequence shown in SEQ ID NO 270 or 271; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 272; and/or

b) A light chain Variable (VL) region comprising (i) a VL CDR1 comprising the sequence set forth in SEQ ID NO: 273; (ii) VL CDR2 comprising the sequence shown in SEQ ID NO. 274; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 275.

8. The bispecific antibody of claim 4, wherein both the first antibody variable domain and the second antibody variable domain of the heterodimeric protein comprise amino acid modifications at positions 223, 225, and 228 in the hinge region and amino acid modifications at positions 409 or 368 in the CH3 region (SEQ ID NO:279) of human IgG2 (EU numbering scheme).

9. The bispecific antibody of claim 8, further comprising amino acid modifications at one or more of positions 265, 330 and 331 of the human IgG 2.

10. A nucleic acid encoding the antibody of any one of claims 1 to 9.

11. A vector comprising the nucleic acid of claim 10.

12. A host cell comprising the nucleic acid of claim 10.

13. The antibody of any one of claims 1 to 9 for use as a medicament.

14. The antibody of claim 13, wherein the medicament is for treating a CD 70-associated cancer selected from the group consisting of: renal cell carcinoma, glioblastoma, gliomas such as low grade glioma, non-hodgkin's lymphoma (NHL), Hodgkin's Disease (HD), waldenstrom's macroglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

15. A method of treating a subject in need thereof, the method comprising:

a) providing an antibody of any one of claims 1-9; and

b) administering the antibody to the subject.

16. A pharmaceutical composition comprising the antibody of any one of claims 1 to 9.

17. A method of treating a disorder associated with malignant cells expressing CD70 in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody of any one of claims 1 to 9 or the pharmaceutical composition of claim 16.

18. The method of claim 17, wherein the disorder is cancer.

19. The method of claim 18, wherein the cancer is a CD 70-associated cancer selected from the group consisting of: renal cell carcinoma, glioblastoma, gliomas such as low grade glioma, non-hodgkin's lymphoma (NHL), Hodgkin's Disease (HD), waldenstrom's macroglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

20. A method of inhibiting tumor growth or progression in a subject having malignant cells that express CD70, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 16.

21. A method of inhibiting metastasis of a malignant cell that expresses CD70 in a subject, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 16.

22. A method of causing tumor regression in a subject having malignant cells expressing CD70, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 16.

23. A method of producing an antibody, the method comprising culturing the host cell of claim 12 under conditions that result in production of the antibody, and isolating the antibody from the host cell or culture.